CN112930397A - ENPP1 polypeptides and methods of use thereof - Google Patents

Abstract

The present invention includes ENPP1 polypeptides having improved in vivo half-life. The invention further provides ENPP1 polypeptide fusions comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the polypeptide fusion comprises at least one point mutation.

Description

ENPP1 polypeptides and methods of use thereof

Cross Reference to Related Applications

Priority of U.S. provisional patent application No. 62/725,607 filed on 2018, 31, 2018 and U.S. provisional patent application No. 62/830,247 filed on 2019, 4, 5, are claimed in this application in accordance with 35 u.s.c. 119(e), all of which are incorporated herein by reference in their entirety.

Background

The human ectonucleotide pyrophosphatase (ENPP) protein family comprises seven extracellular glycosylated proteins that hydrolyze phosphodiester bonds (i.e., ENPP1-ENPP 7). ENPP is a cell surface enzyme, except ENPP2, which exports ENPP2 to the plasma membrane, but is cleaved by furin and released into the extracellular fluid. ENPP enzymes have high sequence and structural homology, but are shown to cover diverse substrate specificity from nucleotides to lipids.

ENPP1 (also known as PC-1) is a type 2 cell outer membrane-binding glycoprotein that is localized on mineral-depositing matrix vesicles of osteoblasts and chondrocytes and hydrolyzes extracellular nucleotides (mainly ATP) to Adenosine Monophosphate (AMP) and inorganic pyrophosphate (PPi). PPi functions as an effective inhibitor of ectopic tissue mineralization: it prevents future growth of new Hydroxyapatite (HA) crystals by binding to these crystals. ENPP1 produces PPi through hydrolysis of Nucleoside Triphosphates (NTPs), progressive tonic protein (ANK) transports intracellular PPi to the extracellular space, and tissue non-specific alkaline phosphatase (TNAP) removes PPi by direct hydrolysis of PPi to Pi.

Ectopic tissue mineralization is associated with a variety of human diseases, including chronic joint disease and acute fatal neonatal syndrome. To prevent unwanted tissue calcification, a tight balance of factors that promote and inhibit tissue mineralization must be maintained. The balance of extracellular inorganic pyrophosphate (PPi) and phosphate (Pi) is an important regulator of ectopic tissue mineralization. The activity of the three extracellular enzymes TNAP, ANK and ENPP1 tightly controlled the concentration of Pi and PPi in mammals at 1-3mM and 2-3. mu.M, respectively. PPi is a biomineralization modulator that inhibits the formation of basic calcium phosphate from amorphous calcium phosphate.

ENPP1 polypeptides have been shown to be effective in treating certain ectopic tissue calcification diseases. ENPP1-Fc has been shown to reduce systemic arterial calcification in a mouse model of GACI (infant systemic arterial calcification), a severe disease that occurs in infants and is involved in extensive arterial calcification (Albright et al 2015, Nature comm.10006). Fusion proteins of ENPP1 have also been described to treat severe tissue calcification diseases (PCT application publication nos. WO2014/126965 and WO2016/187408), and fusion proteins of ENPP1 that include a bone targeting domain have been described to treat GACI (PCT application publication No. WO/2012/125182).

There is a need in the art for polypeptides that can be used in vivo to treat certain calcific or ossifying diseases. Such polypeptides should have an in vivo half-life that allows for convenient and effective administration of the polypeptide to a subject in need thereof. The present invention satisfies this need.

Disclosure of Invention

The present invention provides ENPP1 polypeptide fusions comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the polypeptide fusions comprise at least one point mutation as described herein. The present invention further provides ENPP1 mutant polypeptides comprising at least one point mutation as described elsewhere herein. The invention further provides polypeptide fusions and/or mutant polypeptides, any of which is expressed from a CHO cell line stably transfected with human ST6 β -galactosamide α -2, 6-sialyltransferase (ST6GAL 1). The invention further provides polypeptide fusions and/or mutant polypeptides, any of which are grown in cell culture supplemented with sialic acid and/or N-acetylmannosamine (1,3,4-O-Bu3 Mannac).

The invention further provides a method of reducing and/or preventing progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion and/or mutant polypeptide of the invention.

The invention further provides a method of reducing and/or preventing progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion and/or mutant polypeptide of the invention.

The present invention further provides a method of reducing and/or preventing the progression of ectopic calcification of soft tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion and/or mutant polypeptide of the invention.

The invention further provides a method of treating, reversing and/or preventing the progression of posterior longitudinal ligament Ossification (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion and/or mutant polypeptide of the invention.

The invention further provides a method of treating, reversing and/or preventing progression of hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion and/or mutant polypeptide of the invention.

The present invention further provides a method of reducing and/or preventing progression of at least one disease selected from: chronic Kidney Disease (CKD), end-stage kidney disease (ESRD), Calcific Uraemic Arteriopathy (CUA), calcification defense, posterior longitudinal ligament Ossification (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, Idiopathic Infantile Arterial Calcification (IIAC), generalized arterial calcification in infants (GACI), and atherosclerotic plaque calcification, comprising administering to a subject a therapeutically effective amount of a polypeptide fusion and/or mutant polypeptide of the invention.

The invention further provides a method of reducing and/or preventing the progression of age-related arteriosclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion and/or mutant polypeptide of the invention.

The invention further provides a method of increasing pyrophosphate (PPi) levels in a subject having a PPi level below the normal level of PPi, comprising administering to the subject a therapeutically effective amount of a polypeptide fusion and/or mutant polypeptide of the invention, whereby after administration the level of PPi in the subject is increased to and maintained at about the same level as the normal level of at least 2 μ Μ.

The invention further provides a method of reducing and/or preventing progression of pathological calcification and/or ossification in a subject having a pyrophosphate (PPi) level below the normal level of PPi, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion and/or mutant polypeptide of the invention, thereby reducing and/or preventing progression of pathological calcification and/or ossification in the subject.

The invention further provides a method of treating ENPP1 deficiency manifested by a decrease in extracellular pyrophosphate (PPi) concentration in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion and/or mutant polypeptide of the invention, thereby increasing the level of PPi in the subject.

Drawings

The following detailed description of illustrative embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings exemplary embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 1 shows the ENPP1 polypeptide (SEQ ID NO:7) contemplated by the present invention. The point mutations were identified with reference to SEQ ID NO. 7, which SEQ ID NO. 7 may also be referred to as "parent compound" or "construct # 770". Labeling protocol amino acid numbering and residues are identified with reference to the numbering scheme shown in SEQ ID NO. 7, followed by amino acids that have replaced residues in SEQ ID NO. 7. For example, the mutation C25N refers to the substitution of asparagine (Asn or N) for the cysteine (Cys or C) at position 25 of SEQ ID NO: 7. Legend: (A) n-terminal signal sequence from hENPP 7; all regions in black (B) represent sequences from hENPP1 without formal domain definition; (C) the auxin B domain of hENPP 1; (D) catalytic domain of hENPP 1; (E) the endonuclease domain of hENPP 1; (F) fc domain from invitvogen plasmid pFUSE-hIgG 1-Fc; (G) a four amino acid linker between hENPP1 and the Fc domain; (H) known glycosylation residues.

Figure 2 shows a histogram summarizing plasma phosphodiesterase activity (as measured using thymidine 5' -p-nitrophenyl monophosphate assay or pNP-TMP assay) following a single injection of certain ENPP1 polypeptides in mice (n-3-5). After 25 hours, phosphodiesterase activity remained elevated in all polypeptides, with higher activity at 75 hours being observed with construct #981 (constructs of interest are listed in the tables included elsewhere herein).

Figure 3 shows in vivo pharmacokinetic data for construct #981 as measured using the pNP-TMP assay to record enzyme activity in mouse plasma samples following subcutaneous injection of the construct. The half-life was estimated to be around 122 hours based on a single subcutaneous bolus of 5 mice. Separate experiments to achieve half-life are described elsewhere herein.

FIG. 4 shows construct #1014, now supplemented with 1,3,4-O-Bu₃Selected in vivo pharmacokinetic data for construct #1014 (indicated as "1014A" in the figure) and construct #981 prepared in CHO cells grown in culture medium of ManNAc. The half-life of the construct can be derived from equation 1, as described elsewhere herein.

FIG. 5 shows three known glycosylation sites in ENPP1, all located in random helical regions: (A) (ii) Asn; (B) n-acetylglucosamine. An additional glycosylation site (identified by surface glycoprotein kinetic measurements) is located in the alpha-helix and is labeled in red. There is a common NLT (Asn Leu Thr) in the PDB labile region, which is not known to be glycosylated. Four additional consensus sequences were found in hENPP1, the glycosylation state of which was unknown. A calcium atom (C); 2 zinc atoms (D); an ATP molecule (E).

Figure 6A shows certain domains of human ENPP1 with loss-of-function mutations known to cause the human disease "systemic arterial calcification in infants" (GACI). In certain embodiments, no glycosylation sites are introduced adjacent to regions with mutations known to cause loss of GACI function (as shown in fig. 6A).

Figure 6B shows the crystal structure of ENPP1, with residues highlighted (and marked with ×) that are known to cause loss of function mutations in GACI. (B) Residues in (b) are located in the catalytic domain and correspond to T238A. As in fig. 5: a calcium atom (C); 2 zinc atoms (D); an ATP molecule (E).

FIGS. 7A-7D show the selection results for phosphodiesterase activity from a high throughput TMP-pNP (p-nitrophenyl thymidine monophosphate) assay for an ENPP1 polypeptide. This is a high throughput assay designed by the inventors to rapidly screen for the glycosylated isoform introduced into construct # 770. The figure illustrates the design and performance of a high throughput screen capable of rapidly assessing the biological efficacy of the parent compound, construct #770, mutant form. The construct number in (#) indicates the original WT clone before the mutation was introduced. The construct numbers in (. -) show clones with possible function-gaining mutations.

Fig. 8A is a band diagram showing the Fc domain of human IgG 1. This domain is fused to the C-terminal part of ENPP1 to improve efficacy. Mutations in the Fc domain were introduced to enhance pH-dependent recycling of FcRn. (A) The site of acid-dependent binding is eliminated. (B) The site of enhanced binding. (C) Cysteine disulfide bond. Fuchsin is a known glycosylation site. Figure 8B shows mutations in the Fc domain of human IgG1 known to enhance pH-dependent recycling of FcRn.

Fig. 9 includes graphs and tables showing the effect of glycosylation on PK (in terms of half-life, in hours) and bioavailability of ENPP1 polypeptides. All mutated PKs were comparable to those in construct # CC07 (770B). Further, construct #951 had PK values similar to construct # CC07, but construct #951 grown in a cell line stably transfected with ST6GAL1 (construct #951-ST) showed improved PK and bioavailability. Construct #930 had a similar half-life, but lower bioavailability than construct # CC 07. In contrast, construct #1020 had a higher bioavailability than construct # CC 07. PK and bioavailability data are given in the table, determined as shown in figures 3,4 and 12 and calculated using equation 1.

FIG. 10 includes a graph and table showing the effect of glycosylation and H1064K/N1065F Fc mutation on the half-life (PK in hours) and bioavailability (AUC) of an ENPP1 polypeptide. All constructs containing H1064/N1065 showed improved half-life and AUC values compared to construct # 770B. Notably, constructs #1048 and #1051 correspond to the same cDNA in two different clones, demonstrating the reproducibility of the PK/AUC analysis provided herein. Construct #1064 was also grown in a cell line stably transfected with ST6GAL1 (construct # 1064-ST). Construct #1057 was also grown in a cell line stably transfected with ST6GAL1 ("-ST") (construct #1057-ST) and stably transfected with ST6GAL1And is supplemented with 1,3,4-O-Bu₃-cell line of ManNAc ("-a") (construct # 1057-ST-a). Construct #1089 was identical to construct #1014, but with the addition of a mutation to eliminate potential trypsin cleavage sites. Construct #1014 was also grown in a cell line stably transfected with ST6GAL1, but in this case PK and bioavailability were not improved. PK and bioavailability data are given in the table, determined as shown in figure 3, figure 4 and figure 12 and calculated using equation 1.

Figure 11 includes graphs and tables showing the effect of glycosylation and M883Y/S885T/T887E Fc mutations on PK (in terms of half-life, in hours) and bioavailability of ENPP1 polypeptides. The AUC for construct #1030 was lower than the other constructs, probably due to the S766N mutation. Constructs #981 and #1028 showed an increase in both PK and AUC values when grown in cell lines stably transfected with ST6GAL 1. PK and bioavailability data are given in the table, determined as shown in figure 3, figure 4 and figure 12 and calculated using equation 1.

FIG. 12 includes a set of graphs showing the effect of expressing constructs in CHO cells stably transfected with human α -2,6-ST to produce recombinant biologics with terminal sialic acid residues possessing both α -2,3 and α -2,6 linkages. These cells were called ST6GAL1 cells or ST cells (denoted "-ST"). The figure also shows the presence of sialic acid or the name 1,3,4-O-Bu₃Effect of growth of the construct in ST6GAL1 cells in the case of high-throughput sialic acid precursor of ManNAc (denoted "-a"). PK and bioavailability data are given in the table, determined as shown in figure 3, figure 4 and figure 12 and calculated using equation 1.

FIGS. 13A-13B show the domain of ENPP1 and selected point mutations introduced into the parent compound (SEQ ID NO: 7). This figure identifies specific point mutations introduced into SEQ ID NO 7. Constructs that have been stably transfected into CHO cells stably transfected with human α -2,6-ST are designated "ST". PK and bioavailability data are given in the table, determined as shown in figure 3, figure 4 and figure 12 and calculated using equation 1.

Figure 14 shows the bioavailability (e.g. area under the curve, or AUC) of certain constructs of the invention classified by mutations in the signal sequence (N-terminal region) region.

Figure 15 shows the bioavailability (e.g., area under the curve, or AUC) of certain constructs of the invention classified by endonuclease region mutations.

Detailed Description

In one aspect, the present invention relates to the discovery that certain ENPP1-Fc derivatives have improved half-lives in vivo as compared to ENPP1-Fc polypeptides known in the art.

In one aspect, glycosylation is facilitated to protect the ENPP1-Fc polypeptide from degradation. This is achieved by introducing additional N-glycan consensus sequences onto the outer surface of the predicted tertiary structure under the guidance of a three-dimensional model of ENPP 1.

In another aspect, pH-dependent FcRn-mediated cell recycling is increased by mutating the Fc domain to enhance the affinity of the fusion protein for the neonatal receptor (FcRn).

In another aspect, sialylation of the fusion protein is enhanced by expressing ENPP1-Fc in a CHO cell line stably transfected with human ST6 β -galactosamide α -2, 6-sialyltransferase (also known as ST6GAL 1).

On the other hand, sialic acid capping was enhanced by supplementing the cell culture medium with N-acetylmannosamine (also known as 1,3,4-O-Bu3ManNAc), which is a "high-throughput" precursor of sialic acid.

In certain embodiments, the bioavailability of ENPP1-Fc (C) when administered subcutaneously is substantially improved by enhancing protein sialylation by expressing the biological agent in CHO cells stably transfected with human alpha-2, 6-sialyltransferase_max). In other embodiments, increasing pH-dependent FcRn-mediated cell recycling by manipulation of the Fc domain results in an improvement in biological half-life in vivo. In still other embodiments, combining CHO cells stably transfected with human α -2, 6-sialyltransferase and growing the cells in N-acetylmannosamine results in a significant increase in half-life and/or biological exposure (AUC). In still other embodiments, combining two or more of the methods described herein into a single construct results in a significant increase in half-life and/or biological exposure (AUC).

In certain embodiments, the polypeptides of the invention are more highly glycosylated compared to other ENPP1-Fc polypeptides in the art. In other embodiments, the polypeptides of the invention have a higher affinity for the neonatal orphan receptor (FcRn) as compared to other ENPP1-Fc polypeptides in the art. In still other embodiments, the polypeptides of the invention have a higher half-life in vivo as compared to other ENPP1-Fc polypeptides in the art. In still other embodiments, the kinetic properties of the parent compound (construct #770) are altered such that the change represents a "gain of function" change in the enzyme rate constant. In still other embodiments, the ENPP1-Fc polypeptide of the invention has an in vivo half-life that is at least about 1.5, 2, 2.5, 3,4, 5,6, 7, 8,9, 10, 12, 14, 16, 18, or 20 fold greater than an ENPP-1 polypeptide described in the art. In still other embodiments, the polypeptides of the invention are administered to a subject at a lower dose and/or less frequently than other ENPP1-Fc polypeptides in the art. In still other embodiments, the polypeptide of the invention is administered to the subject once a month, twice a month, three times a month, and/or four times a month. In still other embodiments, less frequent administration of the polypeptides of the invention results in better patient compliance and/or increased efficacy compared to other ENPP1-Fc polypeptides in the art.

In certain embodiments, the ENPP1-Fc polypeptides of the invention can be used to increase pyrophosphate (PPi) levels in subjects with PPi levels below normal (about 2 μ M). In other embodiments, the ENPP1-Fc polypeptide of the invention can be used to reduce or prevent pathological calcification or ossification progression in a subject with lower than normal levels of PPi. In still other embodiments, the ENPP1-Fc polypeptides of the invention are useful for treating an ENPP1 deficiency manifested by a decrease in extracellular PPi concentration in a subject.

In certain embodiments, the steady state level of plasma PPi achieved following administration of a first dose of a construct of the invention is maintained for a period of at least 2 days, at least 4 days, at least one week, or at least one month.

In certain embodiments, a second dose of a construct of the invention is administered to the subject after an appropriate time interval after two days, four days, one week or one month, such that the steady state level of plasma PPi is maintained at a constant or steady state level and does not return to the subject a lower level of PPi prior to administration of the first dose of the construct of the invention.

Without wishing to be bound by theory, it is believed that maintaining the steady-state concentration of plasma PPi at a normal level reduces and/or prevents the progression of pathological calcification and pathological ossification in a subject.

Certain ENPP1 polypeptides, mutants thereof, or mutant fragments thereof, have been previously disclosed in international PCT application publication nos. WO 2012/125182, WO2014/126965, WO2016/187408, and WO 2018/027024, the entire contents of which are incorporated herein by reference.

Reference will now be made in detail to certain embodiments of the disclosed subject matter. Although the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

Values expressed in a range format should be interpreted in a flexible manner throughout the document to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also include individual values (e.g., 1%, 2%, 3%, and 4%) and sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the specified range. Unless otherwise indicated, the statement "about X to Y" has the same meaning as "about X to about Y". Likewise, unless otherwise indicated, the statement "about X, Y or about Z" has the same meaning as "about X, about Y, or about Z".

Definition of

As used herein, each of the following terms has its associated meaning in this section. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, separation science, and organic chemistry are those well known and commonly employed in the art. It should be understood that the order of steps or order of performing certain actions is immaterial so long as the present teachings are still operable. Any use of chapter headings is intended to aid in reading the document and should not be construed as limiting; information related to the chapter title may occur within or outside of that particular chapter. All publications, patents, and patent documents cited herein are incorporated by reference in their entirety as if individually incorporated by reference.

In the present application, where an element or component is considered to be included in and/or selected from a recited list of elements or components, it is to be understood that the element or component may be any one of the recited elements or components and may be selected from two or more of the recited elements or components.

In the methods described herein, the acts may be performed in any order, unless a time or sequence of operations is explicitly recited. Further, specified actions may be performed concurrently, unless explicitly stated to the contrary by the claim language. For example, a claimed act of doing X and a claimed act of doing Y can be performed concurrently in a single operation, and the resulting method would fall within the literal scope of the claimed method.

As used herein, the terms "a", "an" or "the" are intended to include one or more, unless the context clearly indicates otherwise. The term "or" is used to mean a non-exclusive "or" unless otherwise stated. The statement "at least one of a and B" or "at least one of a or B" has the same meaning as "A, B or a and B".

For clarity, the following notation convention is applied to the present disclosure. In any event, any teaching herein that does not follow this convention remains part of the present disclosure and is fully understood in view of the context of the disclosed teachings. Protein symbols are disclosed in non-italic capital letters. By way of non-limiting example, "ENPP 1" refers to a protein. In certain embodiments, if the protein is a human protein, "h" is used before the protein symbol. In other embodiments, if the protein is a mouse protein, "m" is used before the symbol. Thus, human ENPP 1is referred to as "hENPP 1" and mouse ENPP 1is referred to as "mENPP 1". Human gene symbols are disclosed in italic capital letters. As a non-limiting example, the human gene corresponding to the protein hENPP 1is ENPP 1. Discloses a mouse gene symbol, wherein the first letter is upper case, and the other letters are lower case; further, the mouse gene symbols are italicized. As a non-limiting example, the mouse gene that makes the protein mEnpp 1is Enpp 1. Symbols relating to gene mutations are shown in uppercase text.

As used herein, "about" when referring to a measurable value such as an amount, time duration, etc., is intended to encompass variations of ± 20% or ± 10%, in certain embodiments ± 5%, in certain embodiments ± 1%, in certain embodiments ± 0.1% of the specified value, as such variations are suitable for performing the disclosed methods.

A disease or disorder is "alleviated" if the severity of the symptoms of the disease or disorder, the frequency with which the patient experiences such symptoms, or both, is reduced.

As used herein, the terms "alteration," "defect," "variation," or "mutation" refer to a mutation of a gene that affects the function, activity, expression (transcription or translation), or conformation of the polypeptide encoded therein in a cell, including missense and nonsense mutations, insertions, deletions, frameshifts, and premature termination.

As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, as well as immunoreactive portions of intact immunoglobulins.

The "ATP hydrolysis activity" of ENPP1 can be determined by using an ATP cleavage assay. ENPP1 readily hydrolyzes ATP to AMP and PPi. The steady state Michaelis-Menten enzyme constant of ENPP1 was determined using ATP as a substrate. H which can be reacted enzymaticallyPLC analysis demonstrated that ENPP1 cleaves ATP and confirms the identity of the substrate and reaction product by using ATP, AMP, and ADP standards. In the presence of ENPP1, the ATP substrate degraded over time and had an accumulation of the enzyme product AMP. Using different concentrations of ATP substrate, the initial rate (rate gradient) of ENPP1 was derived in the presence of ATP and the data was fitted to a curve to derive the enzymatic rate constant. The kinetic rate constant of NPP 1is K at physiological pH_m144 μ M and k_cat＝7.8s^-1。

As used herein, the term "AUC" refers to the area under the plasma drug concentration-time curve (AUC) and is related to the actual physical exposure to the drug after administration of a dose of the drug. In certain embodiments, the AUC is expressed in mg x h/L. AUC can be used to measure the bioavailability of a drug as the fraction of unaltered drug that is absorbed intact and reaches the site of action or systemic circulation following administration by any route.

AUC can be calculated using either a linear trapezoidal method or a logarithmic trapezoidal method. The linear trapezoidal method uses linear interpolation between data points to calculate AUC. The OGD and FDA require the use of this method and are standards for bioequivalence testing. For a given time interval (t)₁-t₂) AUC can be calculated as follows:

wherein C is₁And C₂Is the time interval (t)₁And t₂) Average concentration of inner.

The log-trapezoidal method uses log interpolation between data points to calculate AUC. This method is more accurate as the concentration decreases, since drug elimination is exponential (which makes it linear on a logarithmic scale). For a given time interval (t)₁–t₂) AUC can be calculated as follows (assuming C₁>C₂)：

As used herein, the term "bioavailability" refers to the extent and rate at which an active moiety (protein, drug, or metabolite) enters the systemic circulation and thus enters the site of action. The bioavailability of the active moiety is determined in large part by the nature of the dosage form, which in part depends on its design and manufacture. Differences in bioavailability between formulations of a given drug or protein can be clinically significant; therefore, it is crucial to know whether a pharmaceutical formulation is equivalent. The most reliable measure of the bioavailability of a drug or protein is the area under the plasma concentration-time curve (AUC). AUC is proportional to the total amount of unaltered drug or therapeutic protein that reaches the systemic circulation. The extent and rate of absorption of a drug or therapeutic protein can be considered bioequivalent if its plasma concentration profiles are substantially superimposable.

The term "bioavailability" of a drug or therapeutic product is defined as the fraction of unaltered drug that is absorbed intact and reaches the site of action or systemic circulation after administration by any route. For intravenous doses of a drug, bioavailability is defined as unity. For drugs administered by other routes of administration, bioavailability is generally less than one. Incomplete bioavailability may be due to a number of factors that can be subdivided into categories of dosage form effects, membrane effects, and application site effects. The half-life and AUC provide information about the bioavailability of a drug or biologic.

As used herein, the term "conservative variation" or "conservative substitution" as used herein refers to the replacement of an amino acid residue by another, biologically similar residue. Conservative variations or substitutions are unlikely to change the shape of the peptide chain. Examples of conservative variations or substitutions include the replacement of one hydrophobic residue with another (e.g., isoleucine, valine, leucine or methionine), or the replacement of one polar residue with another, such as the replacement of arginine with lysine, glutamic with aspartic acid, or glutamine with asparagine.

As used herein, a "construct" of the invention refers to a fusion polypeptide comprising an ENPP1 polypeptide or fragment or site-directed mutant thereof.

A "disease" is a health state of an animal in which the animal is unable to maintain homeostasis, and in which the health condition of the animal will continue to deteriorate if the disease is not ameliorated.

A "disorder" of an animal is a state of health in which the animal is able to maintain homeostasis, but in which the state of health of the animal is adverse compared to the state without the disorder. The disorder does not necessarily lead to a further reduction in the health of the animal if left untreated.

As used herein, the terms "effective amount," "pharmaceutically effective amount," and "therapeutically effective amount" refer to an amount of an agent that is non-toxic but sufficient to provide the desired biological result. The result can be a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In any individual case, the appropriate therapeutic amount can be determined by one of ordinary skill in the art using routine experimentation.

As used herein, the term "ENPP" or "NPP" refers to ectonucleotide pyrophosphatase/phosphodiesterase.

As used herein, the term "ENPP 1 protein" or "ENPP 1 polypeptide" refers to an ectonucleotide pyrophosphatase/phosphodiesterase-1 protein encoded by the ENPP1 gene. The encoded protein is a type II transmembrane glycoprotein and cleaves a variety of substrates, including the phosphodiester bonds of nucleotides and nucleotide sugars and the pyrophosphate bonds of nucleotides and nucleotide sugars. ENPP1 protein has a transmembrane domain and a soluble extracellular domain. The extracellular domain is further subdivided into a somatomedin B domain, a catalytic domain, and a nuclease domain. The sequence and structure of wild-type ENPP 1is described in detail in PCT application publication No. WO2014/126965 to Braddock et al, which is incorporated herein by reference in its entirety.

In the context of functional derivatives of amino acid sequences, the term "functional equivalent" or "functional derivative" refers to a molecule that retains a biological activity substantially similar to the biological activity (function or structure) of the sequence of the ENPP1-Fc construct shown herein. The functional derivatives or equivalents may be natural derivatives or synthetically prepared. Functionally equivalent polypeptides of the invention can also be polypeptides identified using one or more structural and/or sequence alignment techniques known in the art.

Exemplary functional derivatives include amino acid sequences having substitutions, deletions or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The substituted amino acid desirably has similar chemical-physical properties as the substituted amino acid. Desirable similar chemical-physical properties include similarity in charge, bulk, hydrophobicity, hydrophilicity, and the like. Generally, greater than 30% identity between two polypeptides is considered an indication of functional equivalence. Preferably, the functionally equivalent polypeptide of the invention has a degree of sequence identity with the ENPP1-Fc construct of more than 80%. More preferred polypeptides have a degree of identity greater than 85%, 90%, 95%, 98% or 99%, respectively. Methods for determining whether a functional equivalent or functional derivative has the same or similar or higher biological activity as the ENPP1-Fc construct may be determined by using the enzymatic assay described in WO2016/187408 involving ATP cleavage.

As used herein, the term "human ENPP 1" refers to the human ENPP1 sequence as described in NCBI accession No. NP _ 006199. As used herein, the term "soluble human ENPP 1" refers to a polypeptide corresponding to residues 96 to 925 of NCBI accession No. NP _ 006199. The term "enzymatically active" as used herein with respect to ENPP 1is defined as being capable of binding to and hydrolyzing ATP to AMP and PPi and/or binding to and hydrolyzing AP3a to ATP.

As used herein, the term "ENPP 1 precursor protein" refers to ENPP1 having its signal peptide sequence at the N-terminus of ENPP 1. After proteolysis, the signal sequence is cleaved from ENPP1 to provide ENPP1 protein. Signal peptide sequences useful in the present invention include, but are not limited to, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and/or ENPP5 signal peptide sequence.

As used herein, the term "ENPP 1-Fc" refers to ENPP1 recombinantly fused and/or chemically conjugated (including covalent and non-covalent conjugation) to the FcR binding domain of an IgG molecule (preferably, human IgG). In certain embodiments, the C-terminus of ENPP 1is fused or conjugated to the N-terminus of the FcR binding domain.

As used herein, the term "Fc" refers to the human IgG (immunoglobulin) Fc domain. IgG subtypes such as IgG1, IgG2, IgG3, and IgG4 are contemplated as Fc domains.

As used herein, an "Fc region" is a portion of an IgG molecule that is associated with a crystallizable fragment obtained by papain digestion of the IgG molecule. The Fc region comprises the C-terminal halves of the two heavy chains of an IgG molecule linked by disulfide bonds. It has no antigen binding activity, but contains a carbohydrate moiety and binding sites for complement and Fc receptors, including the FcRn receptor. The Fc fragment comprised the entire second constant domain CH2 (residues 231 and 340 of human IgG1 according to the Kabat numbering system) and the third constant domain CH3 (residues 341 and 447). The term "IgG hinge-Fc region" or "hinge-Fc fragment" refers to the region of an IgG molecule consisting of an Fc region (residues 231 and 447) and a hinge region (residues 216 and 230) extending from the N-terminus of the Fc region. The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another portion of an immunoglobulin, i.e., a variable domain having an antigen binding site. The constant domain comprises the CH1, CH2, and CH3 domains of the heavy chain and the CHL domain of the light chain.

As used herein, the term "Fc receptor" refers to a protein found on the surface of certain cells (including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets and mast cells, and the like) that contributes to the protective function of the immune system. Fc receptors bind to antibodies attached to infected cells or invading pathogens. Immunoglobulin Fc receptors (fcrs) are expressed on all hematopoietic cells and play a key role in antibody-mediated immune responses. Binding of the immune complex to FcR activates effector cells, which leads to phagocytosis, endocytosis of IgG-opsonized particles, release of inflammatory mediators, and antibody-dependent cellular cytotoxicity (ADCC). Fc receptors have been described for all types of immunoglobulins: fc γ R for IgG and Fc γ R for neonatal fcr (fcrn), Fc α R, IgD for Fc epsilon R, IgA for IgE and Fc μ R for IgM. All known Fc receptors belong structurally to the immunoglobulin superfamily, except for FcRn and fcesi, which are structurally related to class I major histocompatibility antigens and C-type lectins, respectively (Fc receptors, Neil a. fangera et al, Encyclopedia of Immunology (second edition), 1998).

As used herein, the term "FcRn receptor" refers to the neonatal Fc receptor (FcRn), also known as Brambell receptor, which is a protein encoded by the FCGRT gene in humans. FcRn specifically binds the Fc domain of an antibody. FcRn extends the half-life of IgG and serum albumin by reducing lysosomal degradation in endothelial cells. IgG, serum albumin and other serum proteins are continuously internalized by pinocytosis. Typically, serum proteins are transported from the endosome to the lysosome, where they are degraded. FcRn-mediated transcytosis of IgG across epithelial cells is possible because FcRn binds IgG at acidic pH (<6.5), but does not bind at neutral or higher pH. IgG and serum albumin are bound by FcRn at weakly acidic pH (<6.5) and circulate to the cell surface where they are released at the neutral pH of the blood (> 7.0). In this way, IgG and serum albumin avoid lysosomal degradation.

The Fc portion of IgG molecules is located in the constant region of the heavy chain, particularly in the CH2 domain. The Fc region binds to Fc receptors (FcRn), which are surface receptors for B cells and are also proteins of the complement system. Binding of the Fc region of IgG molecules to FcRn activates receptor-bearing cells and thus the immune system. Fc residues critical for the interaction of mouse Fc-mouse FcRn and human Fc-human FcRn have been identified (Dall' Acqua et al, 2002, J.Immunol.169(9): 5171-80). The FcRn binding domain comprises the CH2 domain (or FcRn binding portion thereof) of an IgG molecule.

As used herein, the term "fragment," as applied to a nucleic acid, refers to a subsequence of a larger nucleic acid. A "fragment" of a nucleic acid can be at least about 15, 50-100, 100-500, 500-1000, 1000-1500 nucleotides, 1500-2500, or 2500 nucleotides (and any integer value therebetween). As used herein, the term "fragment," as applied to a protein or peptide, refers to a subsequence of a larger protein or peptide that may be at least about 20, 50, 100, 200, 300, or 400 amino acids in length (and any integer value therebetween).

As used herein, the term "in vivo half-life" for a protein and/or polypeptide contemplated in the present invention (e.g., an ENPP1 polypeptide comprising an FcRn binding site) refers to the time required to clear half of the amount administered in an animal from the circulatory system and/or other tissues of the animal. When a clearance curve for ENPP1-Fc fusion proteins was constructed as a function of time, the curve was generally biphasic, with a very rapid alpha phase (which represents the equilibrium between the administered molecule in the intravascular and extravascular spaces and depends in part on the size of the molecule) and a longer beta phase (which represents the catabolism of the molecule in the intravascular space). In certain embodiments, the term "in vivo half-life" actually corresponds to the half-life of the molecule in the beta phase.

The term "instructional material", as used herein, includes publications, records, charts, or any other expression medium that can be used to convey the usefulness of the nucleic acids, peptides, and/or compounds of the invention in a kit for identifying or alleviating or treating a variety of diseases or disorders described herein.

"isolated" refers to an alteration or removal from the native state. For example, a nucleic acid or polypeptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is "isolated. An isolated nucleic acid or protein may be present in a substantially purified form, or may be present in a non-natural environment, such as a host cell.

An "oligonucleotide" or "polynucleotide" is a nucleic acid or compound that specifically hybridizes to a polynucleotide that ranges in length from at least 2, in some embodiments, at least 8, 15, or 25 nucleotides, but can be up to 50, 100, 1000, or 5000 nucleotides in length.

As used herein, the term "patient," "individual," or "subject" refers to a human.

As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful in the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient. There are a variety of techniques in the art for administering compounds including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalation, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary and topical administration.

As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not eliminate the biological activity or properties of the compound and is relatively non-toxic, i.e., the material can be administered to an individual without causing adverse biological effects or interacting in a deleterious manner with any of the components contained in the composition.

As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent or encapsulating material, which is involved in carrying or transporting a compound useful in the present invention within or to a patient such that the compound can perform its intended function. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation (including the compounds useful in the present invention) and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and derivatives thereof. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like, that are compatible with the activity of the compounds useful in the present invention, and are physiologically acceptable to a patient. The "pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of the compounds useful in the present invention. Other additional ingredients that may be included in Pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, ed., Mack Publishing co., 1985, Easton, PA), which is incorporated herein by reference.

As used herein, the language "pharmaceutically acceptable salt" refers to salts of the compounds administered prepared from pharmaceutically acceptable non-toxic acids and bases (including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof).

As used herein, the term "Plasma Pyrophosphate (PPi) level" refers to the amount of pyrophosphate present in the plasma of an animal. In certain embodiments, the animal includes rats, mice, cats, dogs, humans, cows, and horses. Due to release from platelets, it is necessary to measure PPi in plasma rather than in serum. There are a number of ways to measure PPi, one of which is by using an enzymatic assay with a modified uridine diphosphate glucose (UDPG) pyrophosphorylase (best and Seegmiller, 1976, Clin. Chim. acta 66: 241-249; Cheung and Suhadolnik, 1977, anal. biochem 83: 61-63). Normal PPi levels in healthy subjects typically range from about 1 μ M to about 3 μ M, and in some cases between 1-2 μ M. Subjects deficient in ENPP1 expression tend to exhibit lower PPi levels in a range of at least 10% below normal, at least 20% below normal, at least 30% below normal, at least 40% below normal, at least 50% below normal, at least 60% below normal, at least 70% below normal, at least 80% below normal, and any combination thereof. In patients with pathological calcific or ossifying disease, plasma PPi levels below 1 μ M, in some cases below detection levels, are found. In some cases, plasma PPi levels in subjects with pathological calcification or ossification disease are below 0.5 μ M (Arterioscler Thromb, Vasc biol.2014, 34(9): 1985-9; Braddock et al 2015, Nat Commun.6: 10006).

As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues joined by peptide bonds, related naturally occurring structural variants, and synthetic, non-naturally occurring analogs thereof.

As used herein, the term "PPi" refers to pyrophosphate.

As used herein, the term "prevent" or "preventing" refers to the absence of a disorder or disease from developing if no disorder or disease occurs, or the absence of further disorder or disease from developing if a disorder or disease has already developed. The ability of a human to prevent some or all of the symptoms associated with a disorder or disease is also contemplated.

As used herein, "sample" or "biological sample" refers to biological material isolated from a subject. The biological sample may comprise any biological material suitable for detecting mRNA, polypeptide, or other marker of a physiological or pathological process in a subject, and may comprise fluid, tissue, cells, and/or non-cellular material obtained from an individual.

As used herein, the term "signal peptide" refers to a sequence of amino acid residues (e.g., ranging from 10 to 30 residues in length) that bind at the amino terminus of a nascent protein of interest during translation of the protein. The signal peptide is recognized by Signal Recognition Particles (SRPs) and cleaved by signal peptidases after endoplasmic reticulum transport (Lodish et al, 2000, Molecular Cell Biology, 4 th edition).

As used herein, "substantially purified" means substantially free of other components. For example, a substantially purified polypeptide is a polypeptide that has been separated from other components with which it is normally associated in its naturally occurring state. Non-limiting embodiments include a purity of 95%, a purity of 99%, a purity of 99.5%, a purity of 99.9%, and a purity of 100%.

As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent, i.e., a compound useful in the present invention (alone or in combination with another agent) to a patient, or to an isolated tissue or cell line from a patient (e.g., for diagnostic or ex vivo applications), who has a disease or disorder, symptoms of a disease or disorder, or the likelihood of developing a disease or disorder, with the intent to treat, cure, alleviate, alter, remedy, ameliorate, improve, or affect the disease or disorder, symptoms of a disease or disorder, or the likelihood of developing a disease or disorder. Such treatments can be specifically tailored or modified based on knowledge gained from the pharmacogenomics field.

A "vector" is a composition of matter that includes an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

As used herein, the term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. Wild-type genes are most common in the human population and are therefore arbitrarily designed as "normal" or "wild-type" gene forms. In contrast, the term "modified" or "mutant" refers to a gene or gene product that exhibits an alteration in sequence and/or functional properties (i.e., altered characteristics) as compared to the wild-type gene or gene product. Naturally occurring mutants can be isolated; it is identified by the fact that it has altered properties, including altered nucleic acid sequences, compared to the wild-type gene or gene product.

The range is as follows: throughout this disclosure, various aspects of the invention may be presented in a range format. It is to be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have explicitly disclosed all the possible sub-ranges within that range as well as individual numerical values. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range such as 1, 2, 2.7, 3,4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Polypeptides

In one aspect, the invention provides ENPP1-Fc polypeptides. The invention contemplates that the polypeptides of the invention can have one or more of the mutations described herein.

In certain embodiments, the ENPP1 polypeptide includes at least one mutation in a signal sequence as set forth in fig. 13A and/or fig. 13B. In certain embodiments, the mutation is selected from the group consisting of C25N, K27T, and V29N associated with SEQ ID NO 7. In certain embodiments, the mutation is C25N associated with SEQ ID NO. 7. In certain embodiments, the mutation is K27T associated with SEQ ID NO. 7. In certain embodiments, the mutation is V29N associated with SEQ ID NO. 7. In certain embodiments, the ENPP1 polypeptide includes at least one mutation selected from C25N/K27T and V29N related to SEQ ID NO: 7.

In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the catalytic region as set forth in fig. 13A and/or fig. 13B. In certain embodiments, the mutation is selected from the group consisting of K369N and I371T related to SEQ ID NO: 7. In certain embodiments, the mutation is K369N associated with SEQ ID NO. 7. In certain embodiments, the mutation is I371T associated with SEQ ID NO 7. In certain embodiments, the ENPP1 polypeptide comprises the mutation K369N/I371T related to SEQ ID NO: 7.

In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the endonuclease domain as set forth in table 1, table 2, table 3, table 4, table 5, figure 6A, figure 13B, figure 14, and/or figure 15. In certain embodiments, the mutation is selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N associated with SEQ ID No. 7. In certain embodiments, the mutation is P534N associated with SEQ ID NO 7. In certain embodiments, the mutation is V536T associated with SEQ ID NO 7. In certain embodiments, the mutation is R545T associated with SEQ ID NO. 7. In certain embodiments, the mutation is P554L associated with SEQ ID NO. 7. In certain embodiments, the mutation is E592N associated with SEQ ID NO. 7. In certain embodiments, the mutation is R741D related to SEQ ID NO 7. In certain embodiments, the mutation is S766N related to SEQ ID NO 7. In certain embodiments, the ENPP1 polypeptide includes at least one mutation selected from the group consisting of P534N/V536T, P554L/R545T, E592N, E592N/R741D, and S766N in association with SEQ ID NO. 7.

In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the junction region as set forth in fig. 13A and/or fig. 13B. In certain embodiments, the mutation is selected from the group consisting of E864N and L866T related to SEQ ID NO. 7. In certain embodiments, the mutation is E864N associated with SEQ ID NO. 7. In certain embodiments, the mutation is L866T associated with SEQ ID NO. 7.

In certain embodiments, the ENPP1 polypeptide includes at least the mutation E864N/L866T associated with SEQ ID NO: 7.

In certain embodiments, the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, wherein the FcRn binding domain comprises any one of the mutations set forth in table 1, table 2, figure 6A, figure 13B, figure 14, and/or figure 15. In certain embodiments, the mutation is selected from M883Y, S885N, S885T, T887E, H1064K, and N1065F associated with SEQ ID No. 7. In certain embodiments, the mutation is M883Y associated with SEQ ID NO. 7. In certain embodiments, the mutation is S885N associated with SEQ ID NO. 7. In certain embodiments, the mutation is S885T associated with SEQ ID NO. 7. In certain embodiments, the mutation is T887E associated with SEQ ID NO. 7. In certain embodiments, the mutation is H1064K associated with SEQ ID NO 7. In certain embodiments, the mutation is N1065F associated with SEQ ID NO. 7. In certain embodiments, the FcRn binding domain comprises at least one mutation selected from the group consisting of S885N, M883Y, M883Y/S885T/T887E, and H1064K/N1065F associated with SEQ ID No. 7.

In certain embodiments, the ENPP polypeptide comprises at least one mutation selected from the group consisting of C25, K27, V29, C25/K27, K369, I371, K369/I371, P534, V536, R545, P554, E592, R741, S766, P534/V536, P554/R545, E592/R741, E864, L866, E864/L866, M883, S885, T887, H1064, N1065, M883/S885/T887, H1064/N1065 associated with SEQ ID NO. 7.

In certain embodiments, the polypeptide comprises at least one mutation selected from the group consisting of S885N, S766N, M883Y/S885T/T887E, E864N/L866T, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H1064K/N1065F, and P534N/V536T/M883Y/S885T/T887E associated with SEQ ID NO. 7.

In certain embodiments, the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, the polypeptide comprising the mutations M883Y, S885T, and T887E associated with SEQ ID NO: 7.

In certain embodiments, the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, the polypeptide comprising the mutations P534N, V536T, M883Y, S885T, and T887E associated with SEQ ID No. 7.

In certain embodiments, the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, the polypeptide comprising the mutations E592N, H1064K, and N1065F associated with SEQ ID No. 7.

In certain embodiments, the polypeptide comprises an ENPP1 mutant polypeptide, wherein the mutant polypeptide comprises an ENPP1 mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N associated with SEQ ID No. 7.

In certain embodiments, the ENPP1 mutant polypeptide includes at least one mutation selected from the group consisting of S766N, P534N/V536T, P554L/R545T, and E592N associated with SEQ ID NO. 7.

In certain embodiments, the polypeptide further comprises an FcRn binding domain of an IgG.

In certain embodiments, the polypeptide comprises a mutation selected from the group consisting of S885N, S766N, M883Y/S885T/T887E, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H1064K/N1065F, and P534N/V536T/M883Y/S885T/T887E associated with SEQ ID NO 7.

In certain embodiments, the polypeptide comprises the S885N mutation in the FcRn binding domain associated with SEQ ID No. 7.

In certain embodiments, the polypeptide comprises the S766N mutation in the ENPP1 mutant polypeptide related to SEQ ID NO. 7.

In certain embodiments, the polypeptide includes mutations M883Y, S885T, and T887E in the FcRn binding domain associated with SEQ ID No. 7.

In certain embodiments, the polypeptide comprises mutations P534N and V536T in the ENPP1 mutant polypeptide and mutations H1064K and N1065F in the FcRn binding domain in relation to SEQ ID No. 7.

In certain embodiments, the polypeptide comprises mutations P554L and R545T in the ENPP1 mutant polypeptide in relation to SEQ ID NO 7.

In certain embodiments, the polypeptide comprises the mutation S766N in the ENPP1 mutant polypeptide and the mutations H1064K and N1065F in the FcRn binding domain in relation to SEQ ID No. 7.

In certain embodiments, the polypeptide comprises the mutation E592N in the ENPP1 mutant polypeptide and the mutations H1064K and N1065F in the FcRn binding domain in relation to SEQ ID NO 7.

In certain embodiments, the polypeptide comprises mutations P534N and V536T in the ENPP1 mutant polypeptide and mutations M883Y, S885T and T887E in the FcRn binding domain in relation to SEQ ID No. 7.

In certain embodiments, the ENPP1 polypeptide lacks a nuclease domain. In other embodiments, the ENPP1 polypeptide is truncated to remove the nuclease domain. In still other embodiments, the ENPP1 polypeptide is truncated to remove the nuclease domain from about residue 524 to about residue 885 relative to SEQ ID No. 1, leaving only the catalytic domain from about residue 186 to about residue 586 relative to SEQ ID No. 1 for maintaining the catalytic activity of the protein.

In certain embodiments, the ENPP1 polypeptide is modified by a segment of the extracellular region of ENPP1 that comprises a peptidase cleavage site after the signal peptide and between the transmembrane and extracellular domains, as compared to SEQ ID NO: 1.

In certain embodiments, the ENPP1 polypeptide is modified by a segment of the extracellular region of ENPP1 comprising a furin cleavage site between the transmembrane and extracellular domains, as compared to SEQ ID NO: 1. In other embodiments, the ENPP1 polypeptide is not modified by a segment of the extracellular region of ENPP1 having a furin cleavage site between the transmembrane and extracellular domains, as compared to SEQ ID NO: 1.

In certain embodiments, the ENPP1 polypeptide is modified by a segment of the extracellular region of ENPP2 comprising a signal peptidase cleavage site as compared to SEQ ID NO: 1. In other embodiments, the ENPP1 polypeptide is not modified by a segment of the extracellular region of ENPP2 comprising a signal peptidase cleavage site as compared to SEQ ID NO: 1.

In certain embodiments, the polypeptide is soluble. In other embodiments, the polypeptide is a recombinant polypeptide. In still other embodiments, the polypeptide comprises an ENPP1 polypeptide that lacks the transmembrane domain of ENPP 1. In still other embodiments, the polypeptide comprises an ENPP1 polypeptide in which the ENPP1 transmembrane domain has been removed (and/or truncated) and replaced with a transmembrane domain of another polypeptide, such as (as a non-limiting example) ENPP2, ENPP5, or ENPP 7.

In certain embodiments, the polypeptide comprises a signal peptide that results in secretion of a precursor of the ENPP1 polypeptide that is proteolytically processed to produce a polypeptide comprising an ENPP1 polypeptide. In other embodiments, the signal peptide is selected from the group consisting of signal peptides of ENPP2, ENPP5, and ENPP 7. In still other embodiments, the polypeptide comprises an ENPP1 polypeptide, the ENPP1 polypeptide comprising the transmembrane domain of ENPP 1; and another polypeptide, such as (as a non-limiting example) ENPP 2. In still other embodiments, the ENPP1 polypeptide comprises a cleavage product of a precursor ENPP1 polypeptide comprising an ENPP2 transmembrane domain. In still other embodiments, the ENPP2 transmembrane domain comprises residues 12-30 of SEQ ID No. 7, which corresponds to IISLFTFAVGVNICLGFTA.

In certain embodiments, the ENPP1 polypeptide is fused at the C-terminus to the Fc domain of human immunoglobulin 1(IgG1), human immunoglobulin 2(IgG2), human immunoglobulin 3(IgG3), and/or human immunoglobulin 4(IgG 4). In other embodiments, the ENPP1 polypeptide is fused at the N-terminus to the Fc domain of human immunoglobulin 1(IgG1), human immunoglobulin 2(IgG2), human immunoglobulin 3(IgG3), and/or human immunoglobulin 4(IgG 4). In still other embodiments, the presence of an IgFc domain improves half-life, solubility, reduces immunogenicity, and increases the activity of an ENPP1 polypeptide.

In certain embodiments, the ENPP1 polypeptide is fused to human serum albumin at the C-terminus. Human serum albumin may be conjugated to the ENPP1 protein through a chemical linker including, but not limited to, a naturally occurring or engineered disulfide bond, or through genetic fusion to ENPP1 or fragments and/or variants thereof.

In certain embodiments, the polypeptide is further pegylated (fused to a poly (ethylene glycol) chain).

In some embodimentsIn (2), k of the polypeptide to the substrate ATP_catA value greater than or equal to about 3.4(± 0.4) s^-1Enzyme^-1Wherein the k is_catDetermined by measuring the ATP hydrolysis rate of the polypeptide.

In certain embodiments, the K of the polypeptide to the substrate ATP_MA value less than or equal to about 2 μ M, wherein K_MDetermined by measuring the ATP hydrolysis rate of the polypeptide.

In certain embodiments, the polypeptide is formulated as a liquid formulation. In other embodiments, the invention provides a dry product form of a pharmaceutical composition comprising a therapeutic amount of a polypeptide of the invention, whereby the dry product can be reconstituted as a solution of the compound in liquid form.

The invention provides a kit comprising at least one polypeptide of the invention, or a salt or solvate thereof, and instructions for using the polypeptide in a method of the invention.

In certain embodiments, the polypeptide lacks a negatively charged bone targeting sequence. In still other embodiments, polyaspartic domains (about 2 to about 20 or more contiguous aspartic acid residues) are non-limiting examples of negatively charged bone targeting sequences. In other embodiments, the polypeptide has a negatively charged bone targeting sequence.

It is to be understood that the ENPP1 polypeptide according to the invention includes not only the native human protein, but also any fragment, derivative, fusion, conjugate or mutant thereof having the ATP hydrolysing activity of the native protein. As used herein in the present disclosure, the phrase "ENPP 1 polypeptide, mutant or mutant fragment thereof" also includes any compound or polypeptide (such as, but not limited to, a fusion protein) comprising an ENPP1 polypeptide, mutant or mutant fragment thereof. The fusion proteins according to the invention are considered to be bioequivalents to ENPP1, but are intended to provide a longer half-life or higher potency due to increased in vivo biological exposure (as judged by "area under the curve" (AUC) or increased half-life in pharmacokinetic experiments).

Vectors and cells

The invention further provides autonomously replicating or integrating mammalian cell vectors comprising a recombinant nucleic acid encoding a polypeptide of the invention. In certain embodiments, the vector comprises a plasmid or a virus. In other embodiments, the vector comprises a mammalian cell expression vector. In still other embodiments, the vector further comprises at least one nucleic acid sequence that directs and/or controls the expression of the polypeptide. In still other embodiments, the recombinant nucleic acid encodes a polypeptide comprising an ENPP1 polypeptide of the invention and a signal peptide, wherein the polypeptide is proteolytically processed after secretion from a cell to produce an ENPP1 polypeptide of the invention.

In yet another aspect, the present invention provides an isolated host cell comprising a vector of the present invention. In certain embodiments, the cell is a non-human cell. In other embodiments, the cell is mammalian. In still other embodiments, the vectors of the invention comprise a recombinant nucleic acid encoding a polypeptide comprising an ENPP1 polypeptide of the invention and a signal peptide. In still other embodiments, the polypeptide is secreted from the cell and then subjected to proteolytic processing to produce the ENPP1 polypeptide of the invention.

Cloning and expression of ENPP1

ENPP1 or ENPP1 polypeptides were prepared as described in US 2015/0359858 a1, which is incorporated herein by reference in its entirety. ENPP 1is a transmembrane protein, which is located on the surface of cells with distinct endodomains. To express ENPP1 as a soluble extracellular protein, the transmembrane domain of ENPP1 can be exchanged with the transmembrane domain of ENPP2, which results in the accumulation of soluble recombinant ENPP1 in the extracellular fluid of baculovirus cultures.

The signal sequence of any other known protein may also be used to target the extracellular domain of ENPP1 for secretion, such as, but not limited to, the signal sequences of immunoglobulin kappa and lambda light chain proteins. Further, the present invention should not be construed as limited to the polypeptides described herein, but also includes any enzymatically active truncated polypeptide comprising the extracellular domain of ENPP 1.

ENPP1 was made soluble by omitting the transmembrane domain. Human ENPP1(SEQ ID NO:1) was modified to express soluble recombinant protein by replacing the human ENPP1 transmembrane region (e.g., residues 77-98) with the corresponding subdomain of human ENPP2(NCBI accession NP-001124335, e.g., residues 12-30). The modified ENPP1 sequence was cloned into a modified pFastbac FIT vector with a TEV protease cleavage site followed by a C-terminal 9-F1IS tag and cloned and expressed in insect cells and both proteins were expressed in a baculovirus system as described previously (Albright et al, 2012, Blood, 120: 4432-.

Production and purification of ENPP1 and ENPP1 fusion proteins

In certain embodiments, a soluble ENPP1 polypeptide, including an IgG Fc domain or enzymatically/biologically active fragment thereof, is effective to treat, reduce, and/or prevent the progression of a disease or disorder contemplated herein. In other embodiments, the soluble ENPP1 polypeptide does not include a bone targeting domain, such as 2-20 consecutive polyaspartic acid residues or 2-20 consecutive polyglutamic acid residues.

To produce soluble recombinant ENPP1 for in vitro use, ENPP1 was fused to the Fc domain of IgG (referred to as "NPP 1-Fc"), and the fusion protein was expressed in a stable CHO cell line. Proteins can also be expressed from HEK293 cells, baculovirus insect cell systems or CHO cells or pichia expression systems using suitable vectors. The protein may be produced in adherent cells or in suspension cells. Preferably, the fusion protein is expressed in CHO cells. To establish a stable cell line, the nucleic acid sequence encoding the ENPP1 construct was cloned into a suitable vector for large scale protein production.

A number of expression systems are known for the production of ENPP1 fusion proteins, including bacteria (e.g., E.coli and Bacillus subtilis), yeasts (e.g., Saccharomyces cerevisiae, Kluyveromyces lactis, and Pichia pastoris), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. The desired protein may be produced in a conventional manner, for example from a coding sequence inserted into the host chromosome or on an episomal plasmid.

The yeast can be transformed with the coding sequence for the desired protein in any usual manner, e.g., electroporation. Methods for transforming yeast by electroporation are disclosed in Becker and Guarente, 1990, Methods enzymol.194: 182. Successfully transformed cells, i.e., cells comprising a DNA construct of the invention, can be identified by well-known techniques. For example, cells resulting from the introduction of an expression construct can be grown to produce the desired polypeptide. Cells can be harvested and lysed and the cells examined for DNA content using methods such as those described in Southern, 1975, J.mol.biol.98:503 and/or Berent et al, 1985, Biotech 3:208 to detect the presence of DNA. Alternatively, antibodies can be used to detect the presence of proteins in the supernatant.

Useful yeast plasmid vectors include pRS403-406 and pRS413-416, and are generally available from Strat:1.gene Cloning Systems, La Jolla, Calif., USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integrating plasmids (Y1p) and incorporate the yeast selectable markers I-11S3, TRP1, LEU2 and IJRA 3. Plasmid pRS413-416 is a yeast centromere plasmid (YCp).

Various methods have been developed to efficiently ligate DNA to a vector via complementary cohesive ends. For example, complementary homopolymer tracts (homo polymer tracks) may be added to the DNA segment for insertion into the vector DNA. The vector and DNA segments are then joined by hydrogen bonding between complementary homopolymer tails to form recombinant DNA molecules

Synthetic linkers containing one or more restriction sites provide an alternative method of ligating DNA segments into vectors. The DNA segment generated by endonuclease restriction digestion is treated with bacteriophage T4 DNA polymerase or escherichia coli DNA polymerase I, which are enzymes that remove the protruding 3 '-single-stranded ends having 3' -5 '-exonuclease activity and fill the recessed 3' -ends with their polymerization activity.

The combination of these activities thus produces blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme capable of catalyzing ligation of the blunt-ended DNA molecules (e.g., bacteriophage T4 DNA ligase). Thus, the product of the reaction is a DNA segment with a polymeric linker sequence at its end. These DNA segments are then cleaved with an appropriate restriction enzyme and ligated into an expression vector that has been cleaved by an enzyme that produces ends compatible with the ends of the DNA segments.

Clones of single stably transfected cells were then established and screened for high expression clones of the desired fusion protein. Screening of single cell clones for ENPP3 protein expression can be accomplished in a high throughput manner in 96-well plates using the synthetase substrate pNP-TMP (Albright et al 2015, nat. Commun.6:10006) as described previously. After identification of high expressing clones by screening, protein production can be accomplished in shake flasks or in bioreactors as described in Albright et al, 2015, nat. Commun.6: 10006.

Purification of ENPP1 can be accomplished using a combination of standard purification techniques known in the art. Examples of which are described above in the production of ENPP3 protein. After purification, ENPP1-Fc was dialyzed to supplement with Zn²⁺And Mg²⁺PBS (PBSplus) -concentrated to between 5 and 7mg/ml and frozen in 200 and 500. mu.l aliquots at-80 ℃. Aliquots were thawed just prior to use and the specific activity of the solution was adjusted to 31.25au/ml (or about 0.7mg/ml, depending on the preparation) by dilution in PBSplus.

Sequence of

1, SEQ ID NO: hENPP1 amino acid sequence

MERDGCAGGGSRGGEGGRAPREGPAGNGRDRGRSHAAEAPGDPQAAASLLAPMDVGEEPLEKAARARTAKDPNTYKVLSLVLSVCVLTTILGCIFGLKPSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQED

2, SEQ ID NO: ENPP2 amino acid sequence

MARRSSFQSCQIISLFTFAVGVNICLGFTAHRIKRAEGWEEGPPTVLSDSPWTNISGSCKGRCFELQEAGPPDCRCDNLCKSYTSCCHDFDELCLKTARGWECTKDRCGEVRNEENACHCSEDCLARGDCCTNYQVVCKGESHWVDDDCEEIKAAECPAGFVRPPLIIFSVDGFRASYMKKGSKVMPNIEKLRSCGTHSPYMRPVYPTKTFPNLYTLATGLYPESHGIVGNSMYDPVFDATFHLRGREKFNHRWWGGQPLWITATKQGVKAGTFFWSVVIPHERRILTILQWLTLPDHERPSVYAFYSEQPDFSGHKYGPFGPEMTNPLREIDKIVGQLMDGLKQLKLHRCVNVIFVGDHGMEDVTCDRTEFLSNYLTNVDDITLVPGTLGRIRSKFSNNAKYDPKAIIANLTCKKPDQHFKPYLKQHLPKRLHYANNRRIEDIHLLVERRWHVARKPLDVYKKPSGKCFFQGDHGFDNKVNSMQTVFVGYGSTFKYKTKVPPFENIELYNVMCDLLGLKPAPNNGTHGSLNHLLRTNTFRPTMPEEVTRPNYPGIMYLQSDFDLGCTCDDKVEPKNKLDELNKRLHTKGSTEAETRKFRGSRNENKENINGNFEPRKERHLLYGRPAVLYRTRYDILYHTDFESGYSEIFLMPLWTSYTVSKQAEVSSVPDHLTSCVRPDVRVSPSFSQNCLAYKNDKQMSYGFLFPPYLSSSPEAKYDAFLVTNMVPMYPAFKRVWNYFQRVLVKKYASERNGVNVISGPIFDYDYDGLHDTEDKIKQYVEGSSIPVPTHYYSIITSCLDFTQPADKCDGPLSVSSFILPHRPDNEESCNSSEDESKWVEELMKMHTARVRDIEHLTSLDFFRKTSRSYPEILTLKTYLHTYESEI

3, SEQ ID NO: hIgG Fc domain, Fc

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

4, SEQ ID NO: hENPP5 protein export signal sequence

MTSKFLLVSFILAALSLSTTFS-Xaa₂₃Xaa₂₄,

Wherein Xaa₂₃Is absent or is L, and

wherein if Xaa₂₃In the absence of Xaa₂₄Is absent, and if Xaa₂₃Is L, then Xaa₂₄Is absent or is Q

5, SEQ ID NO: hENPP7 protein export signal sequence

MRGPAVLLTV ALATLLAPGA GA

6 of SEQ ID NO: hENPP7 protein export signal sequence

MRGPAVLLTV ALATLLAPGA

SEQ ID NO:7：ENPP1-Fc

MRGPAVLLTVALATLLAPGAGAPSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQEDRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Bold: signal sequence

And (2) conventionally: ENPP1 extracellular domain

And (3) underlining: linker sequences

Italic: fc domains

Method

The invention includes a method of reducing or preventing progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of reducing or preventing progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of reducing or preventing ectopic calcification of soft tissue in a subject in need thereof, comprising reducing, ameliorating or preventing vascular calcification, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes methods of reducing or preventing disease progression caused by ENPP1 deficiency. The ENPP1 deficiency is characterized by a reduced level of ENPP1 activity or a defective level of ENPP1 expression in a subject in need thereof (as compared to the level of ENPP1 activity or ENPP1 expression, respectively, in a normal healthy subject), the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of reducing or preventing disease progression caused by lower levels of plasma PPi in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention to increase the subject's plasma PPi to normal (1-3 μ M) or above normal (30-50% above normal), and thereafter maintaining the plasma PPi at constant normal or above normal levels. The method further comprises administering an additional therapeutically effective amount at intervals of two days, three days, one week or one month to maintain the subject's plasma PPi at a constant normal level or above normal level to reduce or prevent the progression of pathological calcification or ossification.

The invention further includes a method of treating, reversing or preventing the progression of posterior longitudinal ligament Ossification (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of treating, reversing or preventing the progression of rickets hypophosphatemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of reducing or preventing progression of at least one disease selected from: chronic Kidney Disease (CKD), end-stage kidney disease (ESRD), calcific uremic arteriolar disease (CUA), calcification defense, posterior longitudinal ligament Ossification (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, Idiopathic Infantile Arterial Calcification (IIAC), generalized arterial calcification in infants (GACI), and atherosclerotic plaque calcification, the method comprising administering to a subject a therapeutically effective amount of a polypeptide of the present invention.

The invention further includes a method of reducing and/or preventing the progression of age-related arteriosclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of reducing or preventing disease progression caused by a deficiency in ENPP1 (e.g., a reduced level of ENPP1 activity and/or a defective level of ENPP1 expression as compared to the level of ENPP1 activity or the level of ENPP1 expression, respectively, in a normal, healthy subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of reducing or preventing disease progression caused by plasma PPi levels below normal in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention to increase and/or maintain the subject's plasma PPi at a level of about 90%, 95%, 100%, 105%, 110%, 120%, 130%, 140%, or 150% of the normal PPi level (about 1-3 μ M). In certain embodiments, the method further comprises further administering a polypeptide of the invention every two days, three days, one week, or one month to maintain plasma PPi levels at about 90%, 95%, 100%, 105%, 110%, 120%, 130%, 140%, or 150% of normal PPi levels, thereby preventing the progression of pathological calcification or ossification.

The invention further includes a method of treating, reversing or preventing the progression of pseudoxanthoma elasticum (PXE) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of treating, reversing or preventing the progression of atherosclerotic plaque calcification in an arterial blood vessel in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of treating, reversing or preventing the progression of osteoarthritis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of treating, reversing or preventing the progression of arteriosclerosis due to premature aging in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of treating, reversing or preventing the progression of X-linked hypophosphatemic rickets (XLH), Hereditary Hypophosphatemic Rickets (HHRH), hypophosphatemic osteopathy (HBD), Autosomal Dominant Hypophosphatemic Rickets (ADHR), and/or autosomal recessive hypophosphatemic rickets in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of treating, reversing or preventing the progression of age-related osteopenia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of treating, reversing or preventing the progression of ankylosing spondylitis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

The invention further includes a method of treating, reversing, or preventing the progression of pediatric sickle cell anemia stroke in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

In certain embodiments, the pathological calcification is selected from Idiopathic Infantile Arterial Calcification (IIAC) and atherosclerotic plaque calcification.

In certain embodiments, the pathological ossification is selected from posterior longitudinal ligament Ossification (OPLL), rickets with hypophosphatemia, and osteoarthritis.

In certain embodiments, the soft tissue calcification is selected from IIAC and osteoarthritis. In other embodiments, the soft tissue comprises atherosclerotic plaque. In still other embodiments, the soft tissue comprises a muscle artery. In still other embodiments, the soft tissue is selected from the group consisting of a joint and a spine. In still other embodiments, the joint is selected from a hand joint and a foot joint. In still other embodiments, the soft tissue is selected from articular cartilage and vertebral disc cartilage. In still other embodiments, the soft tissue comprises a blood vessel. In still other embodiments, the soft tissue comprises connective tissue.

In certain embodiments, the subject is diagnosed with premature aging.

In certain embodiments, the polypeptide of the invention is a secreted product of the ENPP1 precursor protein expressed in mammalian cells. In other embodiments, the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, wherein the ENPP1 precursor protein is proteolytically processed into the polypeptide of the invention. In still other embodiments, in the ENPP1 precursor protein, the signal peptide sequence is conjugated to the N-terminus of the ENPP1 polypeptide. After proteolysis, the signal sequence is cleaved from the ENPP1 precursor protein to provide the ENPP1 polypeptide. In certain embodiments, the signal peptide sequence is selected from the group consisting of an ENPP1 signal peptide sequence, an ENPP2 signal peptide sequence, an ENPP7 signal peptide sequence, and an ENPP5 signal peptide sequence.

In certain embodiments, the polypeptide is administered to the subject acutely or chronically. In other embodiments, the polypeptide is administered to the subject locally, regionally, parenterally, or systemically.

In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.

In certain embodiments, the polypeptide or its precursor protein is administered by at least one route selected from the group consisting of: subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary and topical. In other embodiments, the polypeptide or its precursor protein is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

In certain embodiments, the polypeptide or precursor protein thereof is administered to the subject acutely or chronically. In other embodiments, the polypeptide or its precursor protein is administered to the subject locally, regionally or systemically. In yet another embodiment, the polypeptide or a precursor protein thereof is delivered on an encoded vector, wherein the vector encodes the protein, and which is transcribed and translated from the vector upon administration of the vector to a subject.

One skilled in the art will appreciate that the present invention is not limited to treatment of a disease or disorder after its determination when equipped with the present disclosure including the methods detailed herein. In particular, the symptoms of the disease or disorder need not have been shown to the point of harm to the subject; indeed, there is no need to detect a disease or disorder in a subject prior to administration of a treatment. That is, it is not necessary that a significant pathology of the disease or disorder has occurred before the invention can provide benefit.

Thus, as described more fully herein, the invention includes methods of preventing a disease or disorder in a subject, wherein the polypeptides of the invention can be administered to the subject prior to the occurrence of the disease or disorder, as discussed elsewhere herein, thereby preventing the development of the disease or disorder. In particular, when the symptoms of the disease or disorder have not been shown to the point of harm to the subject; indeed, there is no need to detect a disease or disorder in a subject prior to administration of a treatment. That is, it is not necessary that a significant pathology of the disease or disorder has occurred before the invention can provide benefit. Thus, the invention includes methods for preventing or delaying the onset, or reducing the progression or growth of a disease or disorder in a subject, as the polypeptides of the invention can be administered to the subject prior to detection of the disease or disorder. In certain embodiments, a polypeptide of the invention is administered to a subject with a strong family history of a disease or disorder, thereby preventing or delaying the onset or progression of the disease or disorder.

With the aid of the disclosure herein, one of skill in the art will therefore appreciate that preventing a disease or disorder in a subject includes administering to the subject a polypeptide of the invention as a prophylactic measure against the disease or disorder.

Pharmaceutical compositions and formulations

The invention provides within the methods described herein pharmaceutical compositions comprising a polypeptide of the invention.

Such pharmaceutical compositions are in a form suitable for administration to a subject, or the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. As is well known in the art, the various components of the pharmaceutical composition may be present in the form of physiologically acceptable salts, for example in combination with physiologically acceptable cations or anions.

In embodiments, a pharmaceutical composition for practicing the methods of the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, a pharmaceutical composition for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.

The relative amounts of the active ingredient, pharmaceutically acceptable carrier and any additional ingredients in the pharmaceutical compositions of the invention will vary depending on the identity, size and condition of the subject being treated and further depending on the route of administration of the pharmaceutical composition. For example, the composition can include between about 0.1% and about 100% (w/w) active ingredient.

Pharmaceutical compositions useful in the methods of the invention may be suitably developed for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ocular, intrathecal, intravenous or another route of administration. Other contemplated formulations include engineered (projected) nanoparticles, liposomal preparations, resealed red blood cells containing active ingredients, and immunologically based formulations. One or more routes of administration will be apparent to the ordinarily skilled artisan and will depend upon a number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or later developed in the pharmacological arts. Typically, such methods of manufacture include the step of bringing into association the active ingredient with the carrier or one or more other adjuvant ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single or multiple dosage units.

As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient to be administered to the subject or a convenient fraction of that dose, for example half or one third of that dose. The unit dosage form can be a single daily dose or one of a plurality of daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form for each dose may be the same or different.

Administration/administration

The regimen of administration may affect the constitution of the effective amount. For example, several divided doses and staggered doses may be administered daily or sequentially, or the doses may be continuously infused, or may also be bolus injections. Further, as indicated in the emergency of a therapeutic or prophylactic situation, the dosage of the therapeutic agent may be proportionally increased or decreased. In certain embodiments, administration of a compound of the invention to a subject elevates the subject's plasma PPi to near normal, wherein the normal level of mammalian PPi is 1-3 μ Μ. "near normal" means 0 to 1.2. mu.M or 0-40% lower or higher than normal, 30nM to 0.9. mu.M or 1-30% lower or higher than normal, 0 to 0.6. mu.M or 0-20% lower or higher than normal, 0 to 0.3. mu.M or 0-10% lower or higher than normal.

Administration of the compositions of the invention to a patient, e.g., a mammal, can be carried out using known procedures, at dosages and for periods of time effective to treat the patient's disease or disorder. The effective amount of the therapeutic compound necessary to achieve a therapeutic effect can vary depending on a variety of factors, such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the condition of the disease or disorder, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical arts. Dosage regimens may be adjusted to provide the optimal therapeutic response. The dosage is determined by the biological activity of the therapeutic compound, which in turn depends on the half-life of the therapeutic compound curve and the area under plasma time. The polypeptide according to the invention may be administered at appropriate intervals every 2 days, or every 4 days, or weekly or monthly to achieve continuous levels of plasma PPi close to (1-3 μ M) or higher than (30-50%) the normal level of PPi. Therapeutic doses of the polypeptides of the invention can also be determined based on half-life or the rate of clearance of the therapeutic polypeptide from the body. The polypeptide according to the invention is administered at suitable intervals of time every 2 days, or every 4 days, weekly or monthly to achieve a constant level of enzymatic activity of ENPP 1.

For example, several divided doses may be administered daily or the dose may be reduced proportionally as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dosage range of a therapeutic compound of the invention is about 0.01 to 50mg/kg body weight/day. In some embodiments, an effective dose of a therapeutic compound of the invention ranges from about 50ng to 500ng/kg body weight, preferably 100ng to 300ng/kg body weight. One of ordinary skill in the art will be able to study the relevant factors and determine an effective amount of a therapeutic compound without undue experimentation.

The compound may be administered to the patient frequently several times per day, or may be administered less frequently, such as once per day, once per week, once per two weeks, once per month, or even less frequently, such as once per several months or even once a year or less. It is to be understood that in non-limiting examples, the amount of compound administered daily can be administered daily, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with administration every other day, a dose of 5mg per day may be administered beginning on Monday, the first subsequent dose of 5mg per day on Wednesday, the second subsequent dose of 5mg per day on Friday, and so on. The frequency of dosage will be apparent to the skilled person and will depend on a number of factors, such as, but not limited to, the type and severity of the disease being treated and the type and age of the patient.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

A physician, e.g., a physician, having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start a dose of a compound of the invention for use in a pharmaceutical composition at a lower level than required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

In certain embodiments, the compositions of the present invention are administered to a patient in a dosage ranging from once to five or more times per day. In other embodiments, the compositions of the present invention are administered to a patient at doses ranging from, but not limited to, daily, every second day, every third day to once a week, and every second week. The frequency of administration of the various compositions of the invention will vary from subject to subject depending on a number of factors including, but not limited to, age, the disease or disorder being treated, sex, general health, and other factors. Thus, the invention should not be construed as limited to any particular dosage regimen and precise dosage and the composition administered to any patient is determined by the attending physician taking into account all other factors associated with the patient.

In certain embodiments, the present invention relates to a packaged pharmaceutical composition comprising a container containing a therapeutically effective amount of a compound of the present invention, alone or in combination with a second agent; and instructions for using the compounds to treat, prevent or ameliorate one or more symptoms of a disease or disorder in a subject.

Route of administration

Routes of administration of any of the compositions of the present invention include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (buccal), (transurethral), vaginal (e.g., trans-and perivaginal), nasal (intra) and (transrectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, creams, lozenges, creams, pastes, plasters, lotions, wafers (discs), suppositories, liquid sprays for nasal or oral administration, dry or nebulized formulations for inhalation, compositions and formulations for intravesical administration, and the like. The formulations and compositions useful in the present invention are not limited to the specific formulations and compositions described herein.

Parenteral administration

As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by administration of the pharmaceutical composition with physical disruption of the subject's tissue and by such disruption in the tissue. Thus, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, application of the composition through a surgical incision, application of the composition through a non-surgical wound penetrating tissue, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and renal dialysis infusion techniques.

Additional forms of administration

Additional dosage forms of the invention include those described in U.S. Pat. Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of the invention also include dosage forms described in U.S. patent application nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688 and 20020051820. Additional dosage forms of the invention also include those described in PCT application nos. WO 03/35041, WO 03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO 02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO 98/11879, WO 97/47285, WO 93/18755 and WO 90/11757.

Controlled release formulations and drug delivery systems

Controlled or sustained release formulations of the pharmaceutical compositions of the present invention can be prepared using conventional techniques. In some cases, the dosage form used may be provided with slow or controlled release of one or more of the active ingredients therein, using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes or microspheres, or combinations thereof, to provide the desired release characteristics in varying proportions. The present invention contemplates single unit dosage forms (e.g., tablets, capsules, gelcaps, and caplets) suitable for oral administration that are suitable for controlled release.

In certain embodiments, the formulations of the present invention may be, but are not limited to, short-term, rapidly-counteracting, and controlled formulations such as sustained release, delayed release, and pulsatile release

The term sustained release in its conventional sense refers to a drug formulation that can provide a gradual drug release over an extended period of time, which may, although not necessarily, result in a substantially constant blood level of the drug over an extended period of time. This period of time may be as long as a month or more and is a longer release than the same amount of agent administered in the form of a bolus. For sustained release, the compounds can be prepared with suitable polymeric or hydrophobic materials that provide sustained release properties to the compounds. Thus, the compounds using the methods of the invention may be administered in particulate form (e.g., by injection) or in disc or disc form (by implantation). In some embodiments of the invention, the compounds of the invention are administered to a patient using a sustained release formulation, either alone or in combination with another agent.

The term delayed release is used herein in its conventional sense to refer to a pharmaceutical formulation that provides for the initial release of the drug after some delay following administration of the drug, although not necessarily, it includes delays of from about 10 minutes up to about 12 hours. The term pulsatile release is used herein in its conventional meaning to refer to a pharmaceutical formulation that provides drug release in a manner that produces a plasma profile of the drug pulse following administration of the drug. The term immediate release is used herein in its conventional sense to refer to a pharmaceutical formulation that provides for release of the drug immediately after administration of the drug.

As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration and any or all whole or partial increments thereof after drug administration.

As used herein, rapid offset refers to any time period up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, and any or all whole or partial increments thereof.

Those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims. For example, it is understood that modifications to the reaction and preparation conditions, utilizing art-recognized alternatives and using only routine experimentation, are within the scope of the present application.

It is to be understood that wherever values and ranges are provided herein, all values and ranges subsumed by those values and ranges are to be embraced within the scope of the invention. Moreover, all numbers falling within these ranges, as well as upper or lower limits of the ranges of values, are also contemplated by this application.

The following examples further illustrate various aspects of the invention. However, they are in no way limiting of the teachings or disclosure of the present invention as described herein.

Examples

The invention will now be described with reference to the following examples. These embodiments are provided for illustrative purposes only, and the present invention is not limited to these embodiments, but encompasses all variations apparent from the teachings provided herein.

Method and material

Unless specifically mentioned, construct expression in CHO cells or modified CHO cells with or without supplementation, V, was performed using protocols described elsewhere herein_maxMeasurement, K_m/K_catMeasurement, AUC measurement, half-life measurement.

Generation of ENPP1-Fc mutant constructs

Human NPP1 (human: NCBI accession No. NP 006199) was modified to express soluble recombinant proteins by separatelySubcloning into pFUSE-hlgG1-Fcl or pFUSE-mlgG1-Fcl plasmids (InvivoGen, San Diego CA) and fusion of the recombinant protein with IgG 1. Using a commercially available kit (

Site-directed mutagenesis kit/New England Biolabs) used site-directed mutagenesis to generate constructs from SEQ ID NO 7. The constructs thus generated were sequenced to verify the nucleic acid sequence and then used for protein expression.

Expression of mutant constructs

Stable transfection of the ENPP1-Fc construct was established in CHO K1 cells (Sigma Aldrich, 85051005) under bleomycin (Zeocin)/gentamicin selection and made suitable for suspension growth. Adapted cells were used to inoculate liquid cultures in CD FortiCHO^TMMedium (A1148301, Thermo Fischer) at 37 ℃ and 5% CO₂Grow in shake flasks with stirring at 120rpm at high humidity. The culture was gradually expanded to the desired target volume and then maintained for an additional 2 days to accumulate extracellular protein.

Expression of ENPP1-Fc mutant construct in modified CHO cells

CHO-K1 cells were modified to generate CHO-K1-MOD cells stably expressing human alpha-2, 6-sialyltransferase (alpha-2, 6-ST) enzyme. Stable transfection of the ENPP1-Fc construct was established in CHO K1-MOD cells and the protein was expressed according to the same protocol as described above. Optionally, in some constructs, the cell culture medium of CHO-K1-MOD cells expressing the respective constructs was supplemented with sialic acid or a "high-throughput" precursor of sialic acid referred to as 1,3,4-O-Bu3Mannac to promote higher levels of glycosylation during protein production.

Purification of ENPP1-Fc mutant constructs

The liquid culture was centrifuged at 4300 Xg for 5mm, and the supernatant was filtered through a 0.2 μm membrane and used

3 0.0.11m²

The 30D cartridges (Millipore, Billerica MA) were concentrated by tangential flow. The concentrated supernatant is then purified by a combination of chromatographic techniques in a multi-step process. These techniques are performed sequentially and may include any of the following: affinity chromatography with protein a or protein G, cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, hydrophobic exchange chromatography, High Pressure Liquid Chromatography (HPLC), a precipitation step, an extraction step, a lyophilization step, and/or a crystallization step. Using either of these steps in succession, one of ordinary skill in the art of protein chemistry can purify the composition of matter to homogeneity such that there are no contaminating protein bands on the silver stained gel. The resulting protein samples were then tested using the Pierce LAL Chromogenic Endotoxin quantification Kit (catalog No. 88282) to ensure that all were Endotoxin free.

To quantify the biological impact of clonal optimization, the pharmacodynamic impact of the selected ENPP1-Fc isoform was quantified by measuring plasma PPi concentrations at various time points following a single subcutaneous administration of each isoform.

K_m/K_catMeasurement of

HPLC determined steady state hydrolysis of ATP for the ENPP1 construct. Briefly, the reaction mixture was prepared by adding 20mM Tris (pH 7.4), 150mM NaCl, 4.5nM KCl, 14mM ZnCl₂、1mM MgCl₂And 1mM CaCl₂The enzyme reaction was started by adding 10nM PPi to different concentrations of ATP in the reaction buffer (1). At different time points, 50. mu.l of the reaction solution was removed and quenched with an equal volume of 3M formic acid. The quenched reaction solution was loaded onto a C-18(5m t 250X 4.6mM) column (Higgins Analytical) equilibrated in 5mM ammonium acetate (pH 6.0) solution and eluted with a gradient from 0% to 20% methanol. The substrate and product were monitored by UV absorbance at 259nm and quantified by integration of their corresponding peaks and standard curves.

V_maxMeasurement of

For each mutant prepared, phosphodiesterase activity was analyzed using p-nitrophenyl thymidine 5' -monophosphate (pNP-TMP) (Saunders et al, 2008, mol. cancer ther.7(10): 3352-62; Albright et al, 2015, Nat Commin.6: 10006).

Area under Curve determination

The area under the plasma concentration versus time curve (also referred to as the area under the curve (AUC)) can be used as a method to assess the volume of distribution (V), total clearance for elimination (CL) and bioavailability (F) for extravascular drug delivery. The area under the plasma time curve for each expressed and purified ENPP1-Fc construct was performed using standard equations to determine half-life and bioavailability following a single subcutaneous injection of the biologic, as described in equation 1.

Half-life determination

Half life of drug (t)_1/2) Refers to the time it takes for the plasma concentration or the amount of drug or biological agent in the body to decrease by 50%. The half-life values for each expressed and purified ENPP1-Fc construct were performed following prior art and/or protocols described herein, such as equation 1, which allowed half-life and bioavailability to be determined after a single subcutaneous injection of the biologic.

Drug half-life can be calculated using equation 1, which relates the relationship between systemic fractional concentration of drug administered to a subcutaneous depot in a single injection versus time. Plotting the data as the fraction of drug absorbed (F) as a function of time (t) allows the elimination constant (k) to be determined by fitting the data to the equation for total systemic absorption of drug administered at subcutaneous storage at time t 0_e) And absorption constant (k)_a)。

Example 1: selection and optimization of glycosylation mutations

The AENPP1-Fc construct was mutated to introduce putative additional glycosylation sites and/or to increase Fc affinity for the neonatal orphan receptor (FcRn). The mutations tested are illustrated elsewhere herein, and the specific constructs discussed are illustrated below.

Improvements in the pharmacokinetic properties of ENPP1-Fc were sought by introducing additional N-linked glycosylation sites and enhancing pH-dependent recycling of the fusion protein. As a guide for the selection of additional N-linked glycosylation sites, an electron density map from X-ray diffraction of mouse ENPP1 crystals was used, revealing 4 glycosylation sites in ENPP 1. These sites are hypothesized to be present in highly homologous human ENPP1, and in addition, human ENPP1 contains an additional 4N-linked glycosylation consensus sequences, the glycosylation state of which is unknown (fig. 6B).

To identify the regions of ENPP1 suitable for hyperglycosylation that did not adversely affect catalytic activity, a combination of structural models, clinical data and genetic data of ENPP1 in GACI patients was used. First, an N-linked glycosylation consensus sequence was identified in ENPP2, and sequences that readily allow the introduction of glycosylation sites by altering individual adjacent residues were evaluated. The ENPP2-7 was then structurally modeled using standard software to traverse the mouse ENPP1 structure (PDB ID code 4 GTW). The proposed positions of glycosylation sites were compared to the known position of the inactivated ENPP1 mutation in GACI (fig. 6A-6B) and the position of disulfide bonds in the enzyme. If the proposed spatial position of the glycosylation site is predicted to interfere with either, the site is discarded. These modeling studies led to the identification of potential sites for 53 additional N-linked glycosylation programs that could be readily introduced into ENPP1 without the expectation of disrupting protein folding or enzymatic activity (fig. 7, 13 and 14).

Additional N-linked glycosylation consensus sequences were then introduced into human ENPP1-Fc (h ENPP1-Fc, construct #770) by site-directed mutagenesis. This protein was transiently expressed in CHO cells in 96-well plates and the extracellular supernatant of each clone was screened for enzymatic activity in triplicate in a high throughput assay using pNP-TMP as the chromogenic substrate as described in the methods (fig. 7A-7D). The rate of hydrolysis of pNP-TMP was equal to or better than construct #770 (FIGS. 7A-7D) among 10 of the 53 possible ENPP1-Fc isoforms, and these 10 glycoforms were selected for combinatorial optimization with each other and the IgG1 Fc domain, as described below.

FcRn is the primary homeostatic regulator of human IgG1 Fc serum half-life, while mutations in the Fc domain that enhance pH-dependent interaction of Fc with FcRn prolong the circulating half-life of biological antibodies. The effect of two Fc mutations, H433K/N434F (hereinafter referred to as HN mutation) and M242Y/S254T/T246E (hereinafter referred to as MST mutation), reported to enhance pH-dependent recycling was examined herein (FIGS. 8A-8B). Either of the two variants of the Fc domain was randomly combined with one or more of the 10 ENPP1-Fc glycoforms demonstrating acceptable hydrolysis rates, thereby creating 12 additional ENPP1-Fc clones (table 3). Some of these clones were selected to test the effect of multiple glycoforms on ENPP1-Fc pharmacokinetics, where two spatially distinct putative glycosylation sites on different protein domains were selected to enhance potential glycan shielding on protein surface area (table 3; constructs #1057, #1064, #1014, # 1040). Other clones were tested for the effect of the Fc mutation on the pK properties alone or only in the presence of a single additional putative glycosylation (table 3; constructs #981 and #1051, respectively).

Example 2: expression Using CHO cell lines and growth conditions

Among recombinantly produced proteins, non-human Chinese Hamster Ovary (CHO) cells are widely used in the production of biologics due to the similarity of CHO and human glycosylation patterns. However, there is a glycosylation difference between the two, most notably the terminal sialic acid residues of human N-linked glycans have both α -2,3 and α -2,6 linkages, whereas CHO cells contain only α -2,3 linkages.

To test whether the terminal sialylation differences between CHO and human cells affected PK and bioavailability in this system, a CHO cell line stably expressing human alpha-2, 6-sialyltransferase (alpha-2, 6-ST) was established as the host and this clone was used to produce 7 ENPP1 isoforms to compare the effect of the alpha-2, 6 bond on PK and bioavailability in various constructs (Table 5; construct numbering ending with '-ST'). To explore the effect of growth conditions on PK and bioavailability, cells stably transfected with the selected ENPP1-Fc isoform (both CHO K1 cells and CHO K1 cells stably transfected with human α -2,6-ST) were supplemented with a "high-throughput" precursor of sialic acid, called 1,3, during protein production,4-O-Bu₃ManNAc (table 5).

The ENPP1-Fc isoform was purified to homogeneity using the same purification protocol, and the Michaelis-Menton enzyme rate constants and pharmacokinetic properties were determined as described elsewhere herein. Finally, the pharmacodynamic effects of the selected ENPP1-Fc isoform were quantified by measuring plasma PPi concentrations at various time points following a single subcutaneous administration of each isoform.

Example 3: additional pharmacokinetic effects of N-linked glycosylation sites

Addition of N-linked glycosylation sites to glycoform using the in silico predictions and HTS methods described above significantly increased mouse exposure to ENPP1-Fc in vivo — 4-fold in construct # 1020. The size of the ENPP1-Fc isoform in table 2 was compared by SDS-PAGE gels to determine which sequence variations resulted in increased glycosylation and showed that the increase in molecular weight was consistent with the addition of glycosylation. To determine whether sequence changes in construct #1020 successfully introduced glycosylation, MALDI-TOF was used, which also confirmed the presence of glycosylation at these sites.

Example 4: pharmacokinetic Effect of the Fc IgG1 mutation (FIGS. 10-11)

Antibodies in the Fc domain containing mutations that enhance their affinity for FcRn and increase pH-dependent antibody recycling have never been used in therapeutic enzymes fused to the Fc domain. Some Fc mutations successfully increased the affinity of the Fc domain for the FcRn receptor, but resulted in poor PK properties in the antibody PK in vivo, while others were shown to enhance PK properties in vivo.

To determine whether similar Fc changes enhanced the PK properties of the enzyme fusion protein, two specific IgG1 mutations in the Fc previously used for biological antibodies were investigated — H433K/N434F and M242Y/S254T/T246E. Generally, the M242Y/S254T/T246E mutation was found to be superior to H433K/N434F in improving the properties of ENPP 1-Fc. For example, construct #981, having only the M242Y/S254T/T246E mutation compared to construct #770, increased the half-life by 3.3-fold and AUC by 5.8-fold. In contrast, the construct with the H433K/N434F mutant achieved a more modest half-life increase of between 1.2-1.7 fold with multiple ENPP1 mutations.

Example 5 influence of host cells and growth conditions

Expression of proteins in CHO cells stably transfected with human α -2,6-ST has been successfully used to produce recombinant biologics with terminal sialic acid residues possessing both α -2,3 and α -2,6 linkages, reportedly with increased and decreased PK properties depending on the biologics.

To determine whether the α -2,6 linkage affected the PK properties of ENPP1-Fc, the in vivo exposure (AUC) and half-life of the 7 ENPP1-Fc isoforms produced in either CHOK1 cells or CHOK1 cells stably transfected with human α -2,6-ST were directly compared (Table 4). The general trend of production of biologicals in CHOK1 cells stably transfected with human α -2,6-ST is beneficial. The strongest effect was noted in the exposure of organisms to drug (AUC), indicating a 1.7-4.6 fold increase in AUC in the responding isoform (constructs #1057, #1028, #951, #930 and # 981). Another trend is that AUC affected more in the lower initial AUC isoforms (constructs #951 and # 1057). However, the effect in the longer lasting isoforms (constructs #1028 and #981) was considerable, producing AUC values 8-10 times greater than the parent compound produced in CHOK1 cells.

The effect of the alpha-2, 6 linkage on half-life was modest, increasing by 20-30% in the responding construct. To understand the different effects of the α -2,6 linkage on AUC and half-life eggs, the changes in protein activity versus time for the isoforms produced in CHO k1 cells and 1078 cells were compared.

Example 6: pharmacokinetic effects of growth Using high throughput sialic acid precursors

To determine the effect of growth conditions on PK properties, the media of selected clones were supplemented with "high throughput" sialic acid precursor 1,3,4-O-Bu₃ManNAc or sialic acid itself. CHOK1 cells were supplemented with 1,3,4-O-Bu₃ManNAc hardly improved the PK properties of ENPP1-Fc, but the effect on half-life and AUC was remarkable when the biologicals were produced in CHOK1 cells stably transfected with human α -2,6-ST (figure 12 and table 4). For example, with 1,3,4-O-Bu₃ManNAc complementation of construct #1014Cell culture media from CHOK1 cells had little effect on enhancing AUC and appeared to reduce isoform half-life. In contrast, when 1,3,4-O-Bu is added₃The effect was more pronounced when ManNAc was added to the cell culture medium of construct #1057 produced in CHOK1 cells stably transfected with α -2, 6-ST. And in the absence of supplemental 1,3,4-O-Bu₃These effects produced a 4-fold and 2-fold net increase in AUC and half-life compared to the same isoform produced in CHOK1 grown in culture medium in ManNAc (fig. 12 and table 4).

Table 1.

Table 2: the effect of additional N-linked glycosylation on Pharmacokinetics (PK).

Table 3: effect of Fc mutation on Pharmacokinetics (PK).

Table 4: influence of cell lines and mutations on Pharmacokinetics (PK). Preparing those constructs labeled "-ST" using a modified CHO cell line stably transfected with human α -2, 6-sialyltransferase (α -2, 6-ST); this increased the amount of sialylation of the fusion protein when compared to the fusion protein expressed in a normal CHO cell line. Enhanced sialylation of the construct resulted in improved AUC and half-life values.

*: constructs transfected into CHO-K1 cells

**: constructs transfected into CHO-K1-MOD cells

Table 5: influence of sialic acid supplementation on Pharmacokinetics (PK). Preparing those constructs labeled "-ST" using a modified CHO cell line stably transfected with human α -2, 6-sialyltransferase (α -2, 6-ST); this increased the amount of sialylation of the fusion protein when compared to the fusion protein expressed in a normal CHO cell line. Those constructs labelled "-A" were supplemented with 1,3,4-O-Bu during protein production₃ManNAc (a "high-throughput" precursor of sialic acid) in culture medium.

Table 6: list of polypeptides and corresponding mutants

Table 7: list of mutants in ENPP1 polypeptide

Mutated residues	Mutated residues
		C25N	P558N
K27T	E560T
		V29N	E591N
E115N	E592K
		P117T	E592N
P125T	P643T
		A276N	S645T
L278T	S765N
		D285N	S766N
R287T	S885N
		Y364T	R741A
K369N	V793N
		I371T	H794S
H409T	G795T
		P448L	G795N
S449T	H797T
		P521L	E864N
V522T	L866T
		V522N	H1064K
K526N	N1065K
		P528T	M883Y
P534N	S885T
		V536T	T887E
P543L	M1059L
		R544T	N1065S
R545T	I884A
		G548T	H941A
P554H	H1066A
		P554L

The enumerated embodiments:

the following exemplary embodiments are provided, and their numbering should not be construed as specifying the importance level.

Embodiment 1 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the Fc region comprises at least one mutation selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F, associated with SEQ ID NO: 7.

Embodiment 2 provides a polypeptide fusion according to embodiment 1, wherein the Fc region comprises at least one mutation selected from the group consisting of S885N, M883Y, M883Y/S885T/T887E, and H1064K/N1065F associated with SEQ ID NO: 7.

Embodiment 3 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of C25N, K27T and V29N associated with SEQ ID NO: 7.

Embodiment 4 provides a polypeptide fusion according to embodiment 3, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of C25N/K27T and V29N related to SEQ ID NO: 7.

Embodiment 5 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of K369N and I371T related to SEQ ID NO: 7.

Embodiment 6 provides a polypeptide fusion according to embodiment 5, wherein the ENPP1 polypeptide comprises at least the mutation K369N/I371T related to SEQ ID NO: 7.

Embodiment 7 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N associated with SEQ ID NO: 7.

Embodiment 8 provides a polypeptide fusion according to embodiment 7, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of P534N/V536T, P554L/R545T, E592N, E592N/R741D, and S766N in relation to SEQ ID No. 7.

Embodiment 9 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of E864N and L866T related to SEQ ID NO: 7.

Embodiment 10 provides a polypeptide fusion according to embodiment 9, wherein the ENPP1 polypeptide comprises at least the mutation E864N/L866T related to SEQ ID NO: 7.

Embodiment 11 provides a polypeptide fusion according to any one of embodiments 1-10, comprising at least one mutation selected from the group consisting of C25, K27, V29, C25/K27, K369, I371, K369/I371, P534, V536, R545, P554, E592, R741, S766, P534/V536, P554/R545, E592/R741, E864, L866, E864/L866, M883, S885, T887, H1064, N1065, M883/S885/T887, H1064/N1065 associated with SEQ ID No. 7.

Embodiment 12 provides a polypeptide fusion according to any one of embodiments 1-11, wherein the Fc region is IgG.

Embodiment 13 provides a polypeptide fusion according to any one of embodiments 1-12, comprising at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, S766N and E592N related to SEQ ID No. 7.

Embodiment 14 provides a polypeptide fusion according to any one of embodiments 1-12, comprising at least one mutation selected from the group consisting of S766N, P534N/Y536T, P554L/R545T and E592N related to SEQ ID NO: 7.

Embodiment 15 provides a polypeptide fusion according to any one of embodiments 1-12, comprising at least one mutation selected from the group consisting of S885N, S766N, M883Y/S885T/T887E, E864N/L866T, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H1064K/N1065F, and P534N/V536T/M883Y/S885T/T887E associated with SEQ ID No. 7.

Embodiment 16 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, the polypeptide fusion comprising the mutations M883Y, S885T and T887E associated with SEQ ID NO: 7.

Embodiment 17 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, the polypeptide fusion comprising the mutations P534N, V536T, M883Y, S885T and T887E associated with SEQ ID NO: 7.

Embodiment 18 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, the polypeptide fusion comprising the mutations E592N, H1064K and N1065F associated with SEQ ID No. 7.

Embodiment 19 provides an ENPP1 mutant polypeptide comprising SEQ ID No. 7, wherein the mutant polypeptide comprises a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N related to SEQ ID No. 7.

Embodiment 20 provides a mutant polypeptide according to embodiment 19, wherein the mutant polypeptide comprises at least one mutation selected from the group consisting of S766N, P534N/V536T, P554L/R545T and E592N related to SEQ ID NO: 7.

Embodiment 21 provides a mutant polypeptide according to embodiment 19, comprising a mutation selected from the group consisting of S885N, S766N, M883Y/S885T/T887E, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E37592 84/H1064K/N1065F, and P534N/V536T/M883Y/S885/T887E related to SEQ ID No. 7.

Embodiment 22 provides a mutant polypeptide according to embodiment 19, comprising the S885N mutation associated with SEQ ID NO: 7.

Embodiment 23 provides a mutant polypeptide according to embodiment 19, comprising the S766N mutation associated with SEQ ID NO. 7.

Embodiment 24 provides a mutant polypeptide according to embodiment 19, comprising the mutations M883Y, S885T, and T887E related to SEQ ID NO: 7.

Embodiment 25 provides mutant polypeptides according to embodiment 19, including mutations P534N, V536T, H1064K, and N1065F related to SEQ ID No. 7.

Embodiment 26 provides mutant polypeptides according to embodiment 19, including mutations P554L and R545T related to SEQ ID NO: 7.

Embodiment 27 provides a mutant polypeptide according to embodiment 19, comprising the mutations S766N, H1064K and N1065F related to SEQ ID NO: 7.

Embodiment 28 provides mutant polypeptides according to embodiment 19, including mutations E592N, H1064K, and N1065F related to SEQ ID NO: 7.

Embodiment 29 provides mutant polypeptides according to embodiment 19, including mutations P534N, V536T, M883Y, S885T, and T887E related to SEQ ID NO: 7.

Embodiment 30 provides a polypeptide fusion according to any one of embodiments 1-18 or a mutant polypeptide according to any one of embodiments 19-29 expressed from a CHO cell line stably transfected with human ST6 β -galactosamide α -2, 6-sialyltransferase (also known as ST6GAL 1).

Embodiment 31 provides a polypeptide fusion according to any one of embodiments 1-18 or a mutant polypeptide according to any one of embodiments 19-29 supplemented with sialic acid and/or N-acetylmannosamine (also known as 1,3, 4-O-Bu)₃ManNAc) was grown in cell culture.

Embodiment 32 provides a method of reducing or preventing progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of embodiments 19-31.

Embodiment 33 provides a method of reducing or preventing progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of embodiments 19-31.

Embodiment 34 provides a method of reducing or preventing the progression of ectopic calcification of soft tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of embodiments 19-31.

Embodiment 35 provides a method of treating, reversing or preventing the progression of posterior longitudinal ligament Ossification (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of embodiments 19-31.

Embodiment 36 provides a method of treating, reversing or preventing the progression of rickets from hypophosphatemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of embodiments 19-31.

Embodiment 37 provides a method of reducing or preventing progression of at least one disease selected from the group consisting of: chronic Kidney Disease (CKD), end-stage kidney disease (ESRD), calcific uremic arteriolar disease (CUA), calcification defense, posterior ligamentous Ossification (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, Idiopathic Infantile Arterial Calcification (IIAC), generalized arterial calcification in infants (GACI) and atherosclerotic plaque calcification, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of embodiments 19-31.

Embodiment 38 provides a method of reducing or preventing the progression of age-related arteriosclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of embodiments 19-31.

Embodiment 39 provides the method according to embodiment 32, wherein the pathological calcification is selected from Idiopathic Infantile Arterial Calcification (IIAC) and atherosclerotic plaque calcification.

Embodiment 40 provides a method according to embodiment 33, wherein the pathological ossification is selected from posterior longitudinal ligament Ossification (OPLL), hypophosphatemic rickets and osteoarthritis.

Embodiment 41 provides a method according to embodiment 34, wherein the soft tissue calcification is selected from IIAC and osteoarthritis.

Embodiment 42 provides a method according to embodiment 34, wherein the soft tissue is selected from the group consisting of atherosclerotic plaque, muscular artery, joint, spine, articular cartilage, vertebral disc cartilage, blood vessels, and connective tissue.

Embodiment 43 provides a method of increasing pyrophosphate (PPi) levels in a subject having PPi levels below normal, comprising administering to the subject a therapeutically effective amount of a polypeptide according to the polypeptide fusion of any of embodiments 1-18 and 30-31 or a mutant polypeptide according to any of embodiments 19-31, such that after the administration the level of PPi in the subject is increased to a normal level of at least 2 μ Μ and maintained at about the same level.

Embodiment 44 provides a method of reducing or preventing pathological calcification or ossification progression in a subject having a pyrophosphate (PPi) level below a normal level of PPi, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of embodiments 19-31, thereby reducing or preventing pathological calcification or ossification progression in the subject.

Embodiment 45 provides a method of treating an ENPP1 deficiency manifested by a decreased extracellular pyrophosphate (PPi) concentration in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of embodiments 19-31, thereby increasing the level of PPi in the subject.

Embodiment 46 provides a method according to any one of embodiments 32-45, wherein the polypeptide fusion or mutant polypeptide is a secretion product of an ENPP1 precursor protein expressed in a mammalian cell, wherein the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, wherein the ENPP1 precursor protein is proteolytically processed to produce the ENPP1 polypeptide.

Embodiment 47 provides the method according to embodiment 46, wherein the signal peptide sequence is conjugated to the N-terminus of the ENPP1 polypeptide in the ENPP1 precursor protein.

Embodiment 48 provides the method according to any one of embodiments 46-47, wherein the signal peptide sequence is selected from the group consisting of an ENPP1 signal peptide sequence, an ENPP2 signal peptide sequence, an ENPP7 signal peptide sequence, and an ENPP5 signal peptide sequence.

Embodiment 49 provides a method according to any one of embodiments 32-48, wherein the polypeptide fusion or mutant polypeptide is administered to the subject acutely or chronically.

Embodiment 50 provides a method according to any one of embodiments 32-49, wherein the polypeptide fusion or mutant polypeptide is administered to the subject locally, regionally, parenterally or systemically.

Embodiment 51 provides a method according to any one of embodiments 32-50, wherein the polypeptide fusion or mutant polypeptide is administered to the subject by at least one route selected from the group consisting of: subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary and topical.

Embodiment 52 provides a method according to any one of embodiments 32-51, wherein the polypeptide fusion or mutant polypeptide is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

Embodiment 53 provides a method according to any one of embodiments 32-52, wherein the subject is a mammal.

Embodiment 54 provides a method according to embodiment 53, wherein the mammal is a human.

The respective disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entireties. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be devised by others skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the following claims be interpreted to include all such embodiments and equivalent variations.

Sequence listing

<110> university of yale

P. Starbach

D. Braudouk

<120> ENPP1 polypeptides and methods of use thereof

<130> 047162-7180WO1(00976)

<150> US 62/725,607

<151> 2018-08-31

<150> US 62/830,247

<151> 2019-04-05

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 925

<212> PRT

<213> Intelligent people

<400> 1

Met Glu Arg Asp Gly Cys Ala Gly Gly Gly Ser Arg Gly Gly Glu Gly

1 5 10 15

Gly Arg Ala Pro Arg Glu Gly Pro Ala Gly Asn Gly Arg Asp Arg Gly

20 25 30

Arg Ser His Ala Ala Glu Ala Pro Gly Asp Pro Gln Ala Ala Ala Ser

35 40 45

Leu Leu Ala Pro Met Asp Val Gly Glu Glu Pro Leu Glu Lys Ala Ala

50 55 60

Arg Ala Arg Thr Ala Lys Asp Pro Asn Thr Tyr Lys Val Leu Ser Leu

65 70 75 80

Val Leu Ser Val Cys Val Leu Thr Thr Ile Leu Gly Cys Ile Phe Gly

85 90 95

Leu Lys Pro Ser Cys Ala Lys Glu Val Lys Ser Cys Lys Gly Arg Cys

100 105 110

Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala Ala Cys Val Glu

115 120 125

Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys Ile Glu Pro Glu

130 135 140

His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu Lys Arg Leu Thr

145 150 155 160

Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp Lys Gly Asp Cys

165 170 175

Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys Ser Trp Val Glu

180 185 190

Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro Ala Gly Phe Glu

195 200 205

Thr Pro Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe Arg Ala Glu Tyr

210 215 220

Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser Lys Leu Lys Lys

225 230 235 240

Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr Pro Thr Lys Thr

245 250 255

Phe Pro Asn His Tyr Ser Ile Val Thr Gly Leu Tyr Pro Glu Ser His

260 265 270

Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro Lys Met Asn Ala Ser Phe

275 280 285

Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp Tyr Lys Gly Glu

290 295 300

Pro Ile Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys Ser Gly Thr Phe

305 310 315 320

Phe Trp Pro Gly Ser Asp Val Glu Ile Asn Gly Ile Phe Pro Asp Ile

325 330 335

Tyr Lys Met Tyr Asn Gly Ser Val Pro Phe Glu Glu Arg Ile Leu Ala

340 345 350

Val Leu Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg Pro His Phe Tyr

355 360 365

Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser Gly His Ser Tyr Gly Pro

370 375 380

Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val Asp Gly Met Val

385 390 395 400

Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu His Arg Cys Leu

405 410 415

Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu Gln Gly Ser Cys Lys

420 425 430

Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val Lys Asn Ile Lys

435 440 445

Val Ile Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser Asp Val Pro Asp

450 455 460

Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg Asn Leu Ser Cys

465 470 475 480

Arg Glu Pro Asn Gln His Phe Lys Pro Tyr Leu Lys His Phe Leu Pro

485 490 495

Lys Arg Leu His Phe Ala Lys Ser Asp Arg Ile Glu Pro Leu Thr Phe

500 505 510

Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro Ser Glu Arg Lys

515 520 525

Tyr Cys Gly Ser Gly Phe His Gly Ser Asp Asn Val Phe Ser Asn Met

530 535 540

Gln Ala Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys His Gly Ile Glu

545 550 555 560

Ala Asp Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu Met Cys Asp Leu

565 570 575

Leu Asn Leu Thr Pro Ala Pro Asn Asn Gly Thr His Gly Ser Leu Asn

580 585 590

His Leu Leu Lys Asn Pro Val Tyr Thr Pro Lys His Pro Lys Glu Val

595 600 605

His Pro Leu Val Gln Cys Pro Phe Thr Arg Asn Pro Arg Asp Asn Leu

610 615 620

Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu Asp Phe Gln Thr

625 630 635 640

Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile Lys His Glu Thr

645 650 655

Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu Asn Thr Ile Cys

660 665 670

Leu Leu Ser Gln His Gln Phe Met Ser Gly Tyr Ser Gln Asp Ile Leu

675 680 685

Met Pro Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn Asp Ser Phe Ser

690 695 700

Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe Arg Ile Pro Leu

705 710 715 720

Ser Pro Val His Lys Cys Ser Phe Tyr Lys Asn Asn Thr Lys Val Ser

725 730 735

Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn Ser Ser Gly Ile

740 745 750

Tyr Ser Glu Ala Leu Leu Thr Thr Asn Ile Val Pro Met Tyr Gln Ser

755 760 765

Phe Gln Val Ile Trp Arg Tyr Phe His Asp Thr Leu Leu Arg Lys Tyr

770 775 780

Ala Glu Glu Arg Asn Gly Val Asn Val Val Ser Gly Pro Val Phe Asp

785 790 795 800

Phe Asp Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn Leu Arg Gln Lys

805 810 815

Arg Arg Val Ile Arg Asn Gln Glu Ile Leu Ile Pro Thr His Phe Phe

820 825 830

Ile Val Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr Pro Leu His Cys

835 840 845

Glu Asn Leu Asp Thr Leu Ala Phe Ile Leu Pro His Arg Thr Asp Asn

850 855 860

Ser Glu Ser Cys Val His Gly Lys His Asp Ser Ser Trp Val Glu Glu

865 870 875 880

Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val Glu His Ile Thr

885 890 895

Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val Ser Asp Ile Leu

900 905 910

Lys Leu Lys Thr His Leu Pro Thr Phe Ser Gln Glu Asp

915 920 925

<210> 2

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<212> PRT

<213> Intelligent people

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Met Ala Arg Arg Ser Ser Phe Gln Ser Cys Gln Ile Ile Ser Leu Phe

1 5 10 15

Thr Phe Ala Val Gly Val Asn Ile Cys Leu Gly Phe Thr Ala His Arg

20 25 30

Ile Lys Arg Ala Glu Gly Trp Glu Glu Gly Pro Pro Thr Val Leu Ser

35 40 45

Asp Ser Pro Trp Thr Asn Ile Ser Gly Ser Cys Lys Gly Arg Cys Phe

50 55 60

Glu Leu Gln Glu Ala Gly Pro Pro Asp Cys Arg Cys Asp Asn Leu Cys

65 70 75 80

Lys Ser Tyr Thr Ser Cys Cys His Asp Phe Asp Glu Leu Cys Leu Lys

85 90 95

Thr Ala Arg Gly Trp Glu Cys Thr Lys Asp Arg Cys Gly Glu Val Arg

100 105 110

Asn Glu Glu Asn Ala Cys His Cys Ser Glu Asp Cys Leu Ala Arg Gly

115 120 125

Asp Cys Cys Thr Asn Tyr Gln Val Val Cys Lys Gly Glu Ser His Trp

130 135 140

Val Asp Asp Asp Cys Glu Glu Ile Lys Ala Ala Glu Cys Pro Ala Gly

145 150 155 160

Phe Val Arg Pro Pro Leu Ile Ile Phe Ser Val Asp Gly Phe Arg Ala

165 170 175

Ser Tyr Met Lys Lys Gly Ser Lys Val Met Pro Asn Ile Glu Lys Leu

180 185 190

Arg Ser Cys Gly Thr His Ser Pro Tyr Met Arg Pro Val Tyr Pro Thr

195 200 205

Lys Thr Phe Pro Asn Leu Tyr Thr Leu Ala Thr Gly Leu Tyr Pro Glu

210 215 220

Ser His Gly Ile Val Gly Asn Ser Met Tyr Asp Pro Val Phe Asp Ala

225 230 235 240

Thr Phe His Leu Arg Gly Arg Glu Lys Phe Asn His Arg Trp Trp Gly

245 250 255

Gly Gln Pro Leu Trp Ile Thr Ala Thr Lys Gln Gly Val Lys Ala Gly

260 265 270

Thr Phe Phe Trp Ser Val Val Ile Pro His Glu Arg Arg Ile Leu Thr

275 280 285

Ile Leu Gln Trp Leu Thr Leu Pro Asp His Glu Arg Pro Ser Val Tyr

290 295 300

Ala Phe Tyr Ser Glu Gln Pro Asp Phe Ser Gly His Lys Tyr Gly Pro

305 310 315 320

Phe Gly Pro Glu Met Thr Asn Pro Leu Arg Glu Ile Asp Lys Ile Val

325 330 335

Gly Gln Leu Met Asp Gly Leu Lys Gln Leu Lys Leu His Arg Cys Val

340 345 350

Asn Val Ile Phe Val Gly Asp His Gly Met Glu Asp Val Thr Cys Asp

355 360 365

Arg Thr Glu Phe Leu Ser Asn Tyr Leu Thr Asn Val Asp Asp Ile Thr

370 375 380

Leu Val Pro Gly Thr Leu Gly Arg Ile Arg Ser Lys Phe Ser Asn Asn

385 390 395 400

Ala Lys Tyr Asp Pro Lys Ala Ile Ile Ala Asn Leu Thr Cys Lys Lys

405 410 415

Pro Asp Gln His Phe Lys Pro Tyr Leu Lys Gln His Leu Pro Lys Arg

420 425 430

Leu His Tyr Ala Asn Asn Arg Arg Ile Glu Asp Ile His Leu Leu Val

435 440 445

Glu Arg Arg Trp His Val Ala Arg Lys Pro Leu Asp Val Tyr Lys Lys

450 455 460

Pro Ser Gly Lys Cys Phe Phe Gln Gly Asp His Gly Phe Asp Asn Lys

465 470 475 480

Val Asn Ser Met Gln Thr Val Phe Val Gly Tyr Gly Ser Thr Phe Lys

485 490 495

Tyr Lys Thr Lys Val Pro Pro Phe Glu Asn Ile Glu Leu Tyr Asn Val

500 505 510

Met Cys Asp Leu Leu Gly Leu Lys Pro Ala Pro Asn Asn Gly Thr His

515 520 525

Gly Ser Leu Asn His Leu Leu Arg Thr Asn Thr Phe Arg Pro Thr Met

530 535 540

Pro Glu Glu Val Thr Arg Pro Asn Tyr Pro Gly Ile Met Tyr Leu Gln

545 550 555 560

Ser Asp Phe Asp Leu Gly Cys Thr Cys Asp Asp Lys Val Glu Pro Lys

565 570 575

Asn Lys Leu Asp Glu Leu Asn Lys Arg Leu His Thr Lys Gly Ser Thr

580 585 590

Glu Ala Glu Thr Arg Lys Phe Arg Gly Ser Arg Asn Glu Asn Lys Glu

595 600 605

Asn Ile Asn Gly Asn Phe Glu Pro Arg Lys Glu Arg His Leu Leu Tyr

610 615 620

Gly Arg Pro Ala Val Leu Tyr Arg Thr Arg Tyr Asp Ile Leu Tyr His

625 630 635 640

Thr Asp Phe Glu Ser Gly Tyr Ser Glu Ile Phe Leu Met Pro Leu Trp

645 650 655

Thr Ser Tyr Thr Val Ser Lys Gln Ala Glu Val Ser Ser Val Pro Asp

660 665 670

His Leu Thr Ser Cys Val Arg Pro Asp Val Arg Val Ser Pro Ser Phe

675 680 685

Ser Gln Asn Cys Leu Ala Tyr Lys Asn Asp Lys Gln Met Ser Tyr Gly

690 695 700

Phe Leu Phe Pro Pro Tyr Leu Ser Ser Ser Pro Glu Ala Lys Tyr Asp

705 710 715 720

Ala Phe Leu Val Thr Asn Met Val Pro Met Tyr Pro Ala Phe Lys Arg

725 730 735

Val Trp Asn Tyr Phe Gln Arg Val Leu Val Lys Lys Tyr Ala Ser Glu

740 745 750

Arg Asn Gly Val Asn Val Ile Ser Gly Pro Ile Phe Asp Tyr Asp Tyr

755 760 765

Asp Gly Leu His Asp Thr Glu Asp Lys Ile Lys Gln Tyr Val Glu Gly

770 775 780

Ser Ser Ile Pro Val Pro Thr His Tyr Tyr Ser Ile Ile Thr Ser Cys

785 790 795 800

Leu Asp Phe Thr Gln Pro Ala Asp Lys Cys Asp Gly Pro Leu Ser Val

805 810 815

Ser Ser Phe Ile Leu Pro His Arg Pro Asp Asn Glu Glu Ser Cys Asn

820 825 830

Ser Ser Glu Asp Glu Ser Lys Trp Val Glu Glu Leu Met Lys Met His

835 840 845

Thr Ala Arg Val Arg Asp Ile Glu His Leu Thr Ser Leu Asp Phe Phe

850 855 860

Arg Lys Thr Ser Arg Ser Tyr Pro Glu Ile Leu Thr Leu Lys Thr Tyr

865 870 875 880

Leu His Thr Tyr Glu Ser Glu Ile

885

<210> 3

<211> 227

<212> PRT

<213> Intelligent people

<400> 3

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 4

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> chemical Synthesis

<220>

<221> MOD_RES

<222> (23)..(23)

<223> Xaa23 is absent or L

<220>

<221> MOD_RES

<222> (24)..(24)

<223> if Xaa23 is absent, Xaa24 is absent, and if Xaa23 is L, Xaa24 is absent or Q

<400> 4

Met Thr Ser Lys Phe Leu Leu Val Ser Phe Ile Leu Ala Ala Leu Ser

1 5 10 15

Leu Ser Thr Thr Phe Ser Xaa Xaa

20

<210> 5

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> chemical Synthesis

<400> 5

Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu

1 5 10 15

Ala Pro Gly Ala Gly Ala

20

<210> 6

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> chemical Synthesis

<400> 6

Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu

1 5 10 15

Ala Pro Gly Ala

20

<210> 7

<211> 1078

<212> PRT

<213> Artificial sequence

<220>

<223> ENPP1-Fc

<400> 7

Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu

1 5 10 15

Ala Pro Gly Ala Gly Ala Pro Ser Cys Ala Lys Glu Val Lys Ser Cys

20 25 30

Lys Gly Arg Cys Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala

35 40 45

Ala Cys Val Glu Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys

50 55 60

Ile Glu Pro Glu His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu

65 70 75 80

Lys Arg Leu Thr Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp

85 90 95

Lys Gly Asp Cys Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys

100 105 110

Ser Trp Val Glu Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro

115 120 125

Ala Gly Phe Glu Thr Pro Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe

130 135 140

Arg Ala Glu Tyr Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser

145 150 155 160

Lys Leu Lys Lys Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr

165 170 175

Pro Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr Gly Leu Tyr

180 185 190

Pro Glu Ser His Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro Lys Met

195 200 205

Asn Ala Ser Phe Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp

210 215 220

Tyr Lys Gly Glu Pro Ile Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys

225 230 235 240

Ser Gly Thr Phe Phe Trp Pro Gly Ser Asp Val Glu Ile Asn Gly Ile

245 250 255

Phe Pro Asp Ile Tyr Lys Met Tyr Asn Gly Ser Val Pro Phe Glu Glu

260 265 270

Arg Ile Leu Ala Val Leu Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg

275 280 285

Pro His Phe Tyr Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser Gly His

290 295 300

Ser Tyr Gly Pro Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val

305 310 315 320

Asp Gly Met Val Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu

325 330 335

His Arg Cys Leu Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu Gln

340 345 350

Gly Ser Cys Lys Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val

355 360 365

Lys Asn Ile Lys Val Ile Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser

370 375 380

Asp Val Pro Asp Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg

385 390 395 400

Asn Leu Ser Cys Arg Glu Pro Asn Gln His Phe Lys Pro Tyr Leu Lys

405 410 415

His Phe Leu Pro Lys Arg Leu His Phe Ala Lys Ser Asp Arg Ile Glu

420 425 430

Pro Leu Thr Phe Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro

435 440 445

Ser Glu Arg Lys Tyr Cys Gly Ser Gly Phe His Gly Ser Asp Asn Val

450 455 460

Phe Ser Asn Met Gln Ala Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys

465 470 475 480

His Gly Ile Glu Ala Asp Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu

485 490 495

Met Cys Asp Leu Leu Asn Leu Thr Pro Ala Pro Asn Asn Gly Thr His

500 505 510

Gly Ser Leu Asn His Leu Leu Lys Asn Pro Val Tyr Thr Pro Lys His

515 520 525

Pro Lys Glu Val His Pro Leu Val Gln Cys Pro Phe Thr Arg Asn Pro

530 535 540

Arg Asp Asn Leu Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu

545 550 555 560

Asp Phe Gln Thr Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile

565 570 575

Lys His Glu Thr Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu

580 585 590

Asn Thr Ile Cys Leu Leu Ser Gln His Gln Phe Met Ser Gly Tyr Ser

595 600 605

Gln Asp Ile Leu Met Pro Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn

610 615 620

Asp Ser Phe Ser Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe

625 630 635 640

Arg Ile Pro Leu Ser Pro Val His Lys Cys Ser Phe Tyr Lys Asn Asn

645 650 655

Thr Lys Val Ser Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn

660 665 670

Ser Ser Gly Ile Tyr Ser Glu Ala Leu Leu Thr Thr Asn Ile Val Pro

675 680 685

Met Tyr Gln Ser Phe Gln Val Ile Trp Arg Tyr Phe His Asp Thr Leu

690 695 700

Leu Arg Lys Tyr Ala Glu Glu Arg Asn Gly Val Asn Val Val Ser Gly

705 710 715 720

Pro Val Phe Asp Phe Asp Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn

725 730 735

Leu Arg Gln Lys Arg Arg Val Ile Arg Asn Gln Glu Ile Leu Ile Pro

740 745 750

Thr His Phe Phe Ile Val Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr

755 760 765

Pro Leu His Cys Glu Asn Leu Asp Thr Leu Ala Phe Ile Leu Pro His

770 775 780

Arg Thr Asp Asn Ser Glu Ser Cys Val His Gly Lys His Asp Ser Ser

785 790 795 800

Trp Val Glu Glu Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val

805 810 815

Glu His Ile Thr Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val

820 825 830

Ser Asp Ile Leu Lys Leu Lys Thr His Leu Pro Thr Phe Ser Gln Glu

835 840 845

Asp Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

850 855 860

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

865 870 875 880

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

885 890 895

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

900 905 910

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

915 920 925

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

930 935 940

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

945 950 955 960

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

965 970 975

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

980 985 990

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

995 1000 1005

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

1010 1015 1020

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

1025 1030 1035

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

1040 1045 1050

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

1055 1060 1065

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

1070 1075

Claims (54)

1. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein said Fc region comprises at least one mutation selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F, associated with SEQ ID NO: 7.

2. The polypeptide fusion of claim 1 wherein the Fc region comprises at least one mutation selected from the group consisting of S885N, M883Y, M883Y/S885T/T887E, and H1064K/N1065F associated with SEQ ID NO 7.

3. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of C25N, K27T and V29N associated with SEQ ID NO: 7.

4. The polypeptide fusion of claim 3 wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of C25N/K27T and V29N related to SEQ ID NO: 7.

5. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein said ENPP1 polypeptide comprises at least one mutation selected from the group consisting of K369N and I371T related to SEQ ID NO: 7.

6. The polypeptide fusion of claim 5 wherein said ENPP1 polypeptide comprises at least the mutation K369N/I371T related to SEQ ID NO 7.

7. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D and S766N associated with SEQ ID NO: 7.

8. The polypeptide fusion of claim 7 wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of P534N/V536T, P554L/R545T, E592N, E592N/R741D, and S766N related to SEQ ID NO 7.

9. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein said ENPP1 polypeptide comprises at least one mutation selected from the group consisting of E864N and L866T related to SEQ ID NO: 7.

10. The polypeptide fusion of claim 9 wherein said ENPP1 polypeptide comprises at least the mutation E864N/L866T relative to SEQ ID No. 7.

11. The polypeptide fusion of any one of claims 1,3, 5, 7 and 9 comprising at least one mutation selected from the group consisting of C25, K27, V29, C25/K27, K369, I371, K369/I371, P534, V536, R545, P554, E592, R741, S766, P534/V536, P554/R545, E592/R741, E864, L866, E864/L866, M883, S885, T887, H1064, N1065, M883/S885/T887, H1064/N1065 associated with SEQ ID No. 7.

12. The polypeptide fusion of any one of claims 1,3, 5, 7 and 9 wherein the Fc region is IgG.

13. The polypeptide fusion of any one of claims 1,3, 5, 7 and 9 comprising at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, S766N and E592N related to SEQ ID No. 7.

14. The polypeptide fusion of any one of claims 1,3, 5, 7 and 9 comprising at least one mutation selected from the group consisting of S766N, P534N/Y536T, P554L/R545T and E592N related to SEQ ID No. 7.

15. The polypeptide fusion of any one of claims 1,3, 5, 7 and 9 comprising at least one mutation selected from the group consisting of S885N, S766N, M883Y/S885T/T887E, E864N/L866T, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H1064K/N1065F and P534N/V536T/M883Y/S885T/T887E associated with SEQ ID No. 7.

16. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, said polypeptide fusion comprising the mutations M883Y, S885T and T887E associated with SEQ ID NO: 7.

17. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, said polypeptide fusion comprising mutations P534N, V536T, M883Y, S885T and T887E associated with SEQ ID NO: 7.

18. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, said polypeptide fusion comprising the mutations E592N, H1064K and N1065F associated with SEQ ID NO: 7.

19. An ENPP1 mutant polypeptide comprising SEQ ID No. 7, wherein the mutant polypeptide comprises a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T and E592N related to SEQ ID No. 7.

20. The mutant polypeptide of claim 19, wherein the mutant polypeptide comprises at least one mutation selected from the group consisting of S766N, P534N/V536T, P554L/R545T, and E592N related to SEQ ID No. 7.

21. The mutant polypeptide of claim 19, which comprises a mutation selected from the group consisting of S885N, S766N, M883Y/S885T/T887E, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H1064K/N1065F, and P534N/V536T/M883Y/S885T/T887E associated with SEQ ID No. 7.

22. The mutant polypeptide of claim 19, which comprises the S885N mutation associated with SEQ ID No. 7.

23. The mutant polypeptide of claim 19, which comprises the S766N mutation associated with SEQ ID No. 7.

24. The mutant polypeptide of claim 19, which comprises the mutations M883Y, S885T, and T887E associated with SEQ ID No. 7.

25. The mutant polypeptide of claim 19, which comprises mutations P534N, V536T, H1064K and N1065F related to SEQ ID No. 7.

26. The mutant polypeptide of claim 19, which comprises mutations P554L and R545T associated with SEQ ID NO 7.

27. The mutant polypeptide of claim 19, which comprises the mutations S766N, H1064K and N1065F related to SEQ ID No. 7.

28. The mutant polypeptide of claim 19, which comprises mutations E592N, H1064K, and N1065F associated with SEQ ID No. 7.

29. The mutant polypeptide of claim 19, which comprises mutations P534N, V536T, M883Y, S885T and T887E related to SEQ ID No. 7.

30. The polypeptide fusion of any one of claims 1,3, 5, 7, 9, 16, 17 and 18 or the mutant polypeptide of claim 19 expressed by a CHO cell line stably transfected with human ST6 β -galactosamide α -2, 6-sialyltransferase (also known as ST6GAL 1).

31. The polypeptide fusion of any one of claims 1,3, 5, 7, 9, 16, 17 and 18 or the mutant polypeptide of claim 19 supplemented with sialic acid and/or N-acetylmannosamine (also known as 1,3, 4-O-Bu)₃ManNAc) was grown in cell culture.

32. A method of reducing or preventing pathological calcification progression in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a polypeptide fusion according to any one of claims 1,3, 5, 7, 9, 16, 17 and 18 or a mutant polypeptide according to claim 19.

33. A method of reducing or preventing progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of claims 1,3, 5, 7, 9, 16, 17 and 18 or a mutant polypeptide according to claim 19.

34. A method of reducing or preventing the progression of ectopic calcification of soft tissue in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a polypeptide fusion according to any one of claims 1,3, 5, 7, 9, 16, 17 and 18 or a mutant polypeptide according to claim 19.

35. A method of treating, reversing or preventing the progression of posterior longitudinal ligament Ossification (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of claims 1,3, 5, 7, 9, 16, 17 and 18 or a mutant polypeptide according to claim 19.

36. A method of treating, reversing or preventing the progression of hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of any one of claims 1,3, 5, 7, 9, 16, 17 and 18 or the mutant polypeptide of claim 19.

37. A method of reducing or preventing progression of at least one disease selected from: chronic Kidney Disease (CKD), end stage kidney disease (ESRD), calcific uremic arteriolar disease (CUA), calcification defense, posterior ligamentous Ossification (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, Idiopathic Infantile Arterial Calcification (IIAC), systemic arterial calcification in infants (GACI), and atherosclerotic plaque calcification, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of claims 1,3, 5, 7, 9, 16, 17, and 18 or a mutant polypeptide according to claim 19.

38. A method of reducing or preventing the progression of aging-associated arteriosclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of claims 1,3, 5, 7, 9, 16, 17 and 18 or a mutant polypeptide according to claim 19.

39. The method of claim 32, wherein said pathological calcification is selected from Idiopathic Infant Arterial Calcification (IIAC) and atherosclerotic plaque calcification.

40. The method of claim 33, wherein the pathological ossification is selected from posterior longitudinal ligament Ossification (OPLL), hypophosphatemic rickets, and osteoarthritis.

41. The method of claim 34, wherein the soft tissue calcification is selected from IIAC and osteoarthritis.

42. The method of claim 34, wherein the soft tissue is selected from the group consisting of atherosclerotic plaque, muscular artery, joint, spine, articular cartilage, vertebral disc cartilage, blood vessels, and connective tissue.

43. A method of increasing the level of pyrophosphate (PPi) in a subject having a PPi level below the normal level of PPi, comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of any one of claims 1,3, 5, 7, 9, 16, 17 and 18 or a mutant polypeptide of claim 19, such that after said administration the level of PPi in the subject is increased to and maintained at about the normal level of at least 2 μ Μ.

44. A method of reducing or preventing pathological calcification or ossification progression in a subject having a pyrophosphate (PPi) level below a normal level of PPi, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of any one of claims 1,3, 5, 7, 9, 16, 17, and 18 or a mutant polypeptide of claim 19, thereby reducing or preventing pathological calcification or ossification progression in the subject.

45. A method of treating ENPP1 deficiency manifested by a decrease in extracellular pyrophosphate (PPi) concentration in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of any one of claims 1,3, 5, 7, 9, 16, 17, and 18 or a mutant polypeptide of claim 19, thereby increasing the level of the PPi in the subject.

46. The method of any one of claims 32-38 and 43-45, wherein the polypeptide fusion or mutant polypeptide is a secreted product of an ENPP1 precursor protein expressed in a mammalian cell, wherein the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, wherein the ENPP1 precursor protein is proteolytically processed to produce the ENPP1 polypeptide.

47. The method of claim 46, wherein the signal peptide sequence is conjugated to the N-terminus of the ENPP1 polypeptide in the ENPP1 precursor protein.

48. The method of claim 46, wherein the signal peptide sequence is selected from the group consisting of an ENPP1 signal peptide sequence, an ENPP2 signal peptide sequence, an ENPP7 signal peptide sequence, and an ENPP5 signal peptide sequence.

49. The method of any one of claims 32-38 and 43-45, wherein the polypeptide fusion or mutant polypeptide is administered to the subject acutely or chronically.

50. The method of any one of claims 32-38 and 43-45, wherein the polypeptide fusion or mutant polypeptide is administered to the subject locally, regionally, parenterally or systemically.

51. The method of any one of claims 32-38 and 43-45, wherein the polypeptide fusion or mutant polypeptide is administered to the subject by at least one route selected from the group consisting of: subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary and topical.

52. The method of any one of claims 32-38 and 43-45, wherein the polypeptide fusion or mutant polypeptide is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

53. The method of any one of claims 32-38 and 43-45, wherein the subject is a mammal.

54. The method of claim 53, wherein the mammal is a human.

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